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METHOD FOR ANALYSING THE INTERACTION OF NUCLEOTIDE SEQUENCES IN A
THREE-DIMENSIONAL DNA STRUCTURE
FIELD OF THE INVENTION
The present invention relates to a method for analysing the interaction of
nucleotide
sequences in a three-dimensional DNA structure such as chromatin.
BACKGROUND TO THE INVENTION
A number of recent studies have shown that the genome is organised in a number
of
self-associating domains that are separated by linker regions. These so-called
"topological domains", generally range from 300 kilobasepairs (kb) to 1
megabasepair (1Mb). A topological domain consists of a series of chromatin
loops,
where a loop is defined as bringing two parts of the chromatin in close
proximity
allowing interaction between the regions, although the latter need not be the
case.
These loops are dynamic and dependent on a large number of proteins including
CTCF and cohesion and a series of transcription factors required for the
regulation
of genes within the domain. A number of loops within a domain are thought to
be
purely structural, i.e to enable folding of the genome creating separate
domains;
while other loops have a function in the expression of genes. Loops (chromatin
proximity) of the latter type are frequent within topological domains and much
less
so between chromatin located in different topological domains.
Regulatory DNA elements interact with each other and the genes within a domain
and
form complex interaction networks. Changes within these elements and their
interactions (in addition to mutations in the genes) are responsible for
changes in
gene expression, which in turn is responsible for the differences between
individuals
of a species or causing disease. Thus these elements have become important for
the
diagnosis and treatment of disease. However, these regulatory networks are
still
relatively unknown, although significant effort has recently been put into the
elucidation of their function.
Regulatory elements are short fragments which contain one or more binding
sites for
transcription factors which activate or repress genes. Regulatory elements are
often
located far from their target genes and, although they can be recognized by
the
binding of particular factors such as p300 or chromatin modifications, it is
often not
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clear with which genes they interact. In the spatial organization of the
genome, they
are in close proximity with their target genes. For example in polydactyly,
although the
enhancer affected is located about 1Mbp away from the affected growth factor
gene
Shh on a linear map of the genome it is closely associated with the gene in
the 3D
space of the nucleus.
Although it was already clear that regulatory elements regulate genes by
looping,
chromosome conformation capture (3C) brought a revolution in the field by
allowing
the rapid identification of such interactions. The basic principal of the 3C
technique is
that the close proximity of DNA fragments in the nuclear space can be detected
by
crosslinking followed by restriction enzyme digestion, ligation and
amplification of the
ligated product. A number of 3C types techniques have subsequently been
developed which provide more information about the interactions and the way
the
genes are regulated: 3C/3C-qPCR; 3C-seq/4C-seq; 4C (3C-on-a chip); 5C (3C
carbon copy); and Hi-C.
Each of these methods is associated with various advantages and disadvantages
(Table 1). 3C and 4C techniques are quite laborious, require prior knowledge
of the
locus and are restricted to detecting the interactions from a specific
viewpoint. In
order to analyse several interactions, a number of different viewpoints have
to be
used requiring separate analyses. The 3C and 4C techniques do not yield genome
wide data.
The 5C and HiC techniques are more advanced. 5C is highly demanding in primer
design and allows the analysis of a number of separate interactions, but does
not give
genome wide coverage. HiC is very expensive as it requires a very large amount
of
sequencing in order to analyse the whole genome without offering high
resolution
analysis (normally 40Kbp). The most recent HiC method of analysis uses a new
algorithm and provides a resolution of 10 Kbp. However, it requires an
enormous
amount of sequencing (3.4 billion mapped paired-end reads from 6 biological
replicates). Sequencing on this scale is not available to most research
groups. Also,
the interest very often relates to a specific question involving a limited set
of specific
loci or domains, for example the regions involved in genomic alterations in
disease,
which means that a significant proportion of the sequencing performed by the
HiC
method is superfluous for these applications.
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There is thus a need for an improved method for analysing the interaction of
nucleotide sequences in three-dimensional chromatin structure which does not
suffer
from the above limitations.
Table 1 - Comparison between different chromatin conformation capturing
techniques
Method Applications Advantages Disadvantages
3C-qPCR One-to-one Simple analysis Laborious, requires
knowledge of the locus
and proper controls
3C-seq/4C- One-to-all Allows wide coverage, Restricted to single
seq good resolution, good viewpoint per
signal to noise ratio experiment when
multiplexing several
viewpoints, the analysis
requires extra
bioinformatics expertise
3C-on-chip One-to-all Relatively simple data Poor signal to noise
(4C) analysis ratio, difficult to obtain
genome wide coverage,
analysis requires some
bioinformatics expertise
5C Many-to- Identifies interactions Very laborious, no
many between many individual genome wide coverage,
fragments primer design can be
challenging.
HiC All-to-All Explores the genome wide Very expensive, requires
interactions between all a large sequence effort
individual fragments to obtain sufficient
coverage, -10-40Kbp
resolution, requires
advanced bioinformatics
expertise, repetitive
sequences are excluded
from the analysis
3a
In accordance with an aspect of the present invention, there is provided a
method for analysing the interaction of one or more nucleotide sequence(s)
from one
or more region(s) of interest with other nucleotides sequences in a three-
dimensional
DNA structure, comprising the steps of:
(a) providing a sample of cross-linked DNA;
(b) digesting the cross-linked DNA with a first restriction enzyme to form
cross-linked nucleotide sequences;
(c) ligating the cross-linked nucleotide sequences to ligate the ends of a
nucleotide sequence to another nucleotide sequence;
(d) reversing the cross-linking to form ligated molecules;
(e) fragmenting the ligated molecules from step (d) to form fragmented
molecules;
(f) generating a sample of enriched fragments by hybridising the fragmented
molecules of the one or more nucleotide sequence(s) from one or more region(s)
of
interest from step (e) to one or more oligonucleotide probe(s) complementary
to the
sequences which are within 100bp of the restriction site of the first
restriction enzyme
in the one or more region(s) of interest in order to enrich for the ends of
the nucleotide
sequences that have been ligated to another nucleotide sequence in step (c),
wherein
the one or more oligonucleotide probe(s) is/are spotted on a microarray or
captured
on beads, or present in solution, which are subsequently captured on beads;
and
(g) analysing the nucleotide sequence of the enriched fragments in order to
identify the other nucleotide sequences which interact with the one or more
nucleotide
sequence(s) from one or more region(s) of interest.
1321517.1
Date Recue/Date Received 2022-03-20
4
DESCRIPTION OF THE FIGURES
Figure 1: Overview of the T2C procedure
Isolated cross-linked chromatin is digested and ligated under diluted
conditions to
favour links between restriction fragments in close proximity. After
decrosslinking and
a secondary digestion, the overhangs are repaired followed by adaptor
ligation. The
adaptor contains sequences required for the sequencing method e.g. paired end
IIlumina or optionally a short address sequence. Different addresses would be
used in
different samples to allow multiplexing (hybridisation of different samples to
the same
set of oligonucleotide probes) where the address sequence allows the matching
of a
sequence with the sample it was derived from. The resulting library(ies)
is/are
hybridized to a set of unique oligonucleotide probes on an array or
oligonucleotide
probes in solution that can be captured on beads. The unique oligonucleotide
probes
(green squiggles) are located as close as possible to the first restriction
site. The
hybridized DNA is eluted and contains the library of all interactions from the
selected
area of the genome and is pair-end sequenced on an IIlumina HiSeq2000 followed
by
bionformatical analysis and visualization of the interactions (i.e. sequences
in close
proximity). Vertical black lines depict primary restriction enzyme cleavage
sites.
Orange small vertical lines depict secondary restriction enzyme cleavage
sites.
Figure 2: Comparison of interactions detected by T2C for the human chill p15.5
region with Hi-C data and 4C data
A) Hi-C data generated for IMR90 cells covering the H19/IGF2 region of
interest,
presented at 40Kbp resolution.
B) Interactions observed by T2C in HB2 cells are presented using the same
40kbp
bins as in (A). The overall topological domain pattern observed by the two
methods is
similar.
C) T2C data presented in their actual resolution at fragment level. The colour
bar on
the right gives the frequency of sequence reads for each interaction from a
low (blue)
to a high (yellow) number of reads. The number of reads represents the
frequency of
ligation between two fragments and hence the interaction between those
fragments in
the three dimensional space of the nucleus.
D) 4C interaction data for one viewpoint close to the IGF2 gene in comparison
to the
interactions observed for this particular viewpoint by T2C (fat red line). The
viewpoint
is also indicated in (E), to allow a direct comparison between the methods.
the thin
red lines indicate a number of interaction fragments for ease of comparison.
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Figure 3: Comparison of the compartmentalization and of the interactions for
the 13-
globin locus.
T2C performed in a -2 MB region around the f3-globin locus for mouse primary
erythroid cells (A) and mouse fetal brain cells (B) from E12.5 mice. The
topological
domain patterns between different biological materials appear to be the same
independent of the different number of interactions in the two biological
samples.
Zoom in on the interactions around the p-globin locus for mouse primary
erythroid
cells (C) and mouse fetal brain cells (D) from E12.5 mice. White lines
indicate the
areas of particular interest (like 3'HS P-globin promoter and LCR) in the P-
globin
locus. Interactions between LCR, the P-globin promoter and the 3'HS1 are lost
in
mouse brain cells. All the interactions are normalized to the same colour
code.
The linear representation of the locus is shown at the bottom with the binding
sites of
LDB1 and CTCF in erythroid cells.
Figure 4: Comparison of the interactions of the fragments containing LDB1 or
CTCF
binding sites
A interactions over a -2 MB region around the p-globin locus for fragments
that bind
LDB1 (A), (B) or CTCF (C), (D) for mouse primary erythroid cells (A), (C) and
mouse
fetal brain cells (B), (D) from E12.5 mice. The topological domain around the
p-globin
locus is clearly depicted in the mouse liver cells when compared to mouse
brain cells.
A zoom in presentations of the interaction between LDB1 bound fragments (E),
(F)
and CTCF bound fragments (G), (H). around the f3-globin locus for mouse
primary
erythroid cells (E), (G) and mouse brain cells (F), (H). With white lines
indicate
particular areas of interest (like 3'HS1, the p-globin promoter and the LCR)
in the 13-
globin locus. The fetal liver interactions between LCR, P-globin promoter and
3'HS1
are lost in mouse brain cells. All the interactions are normalized to the same
colour
code. The bottom shows a linear representation of the p-globin locus with the
binding
sites of LDB1 and CTCF in erythroid cells.
Figure 5: The mean, median and the number of interactions for the LDB1 or CTCF
only containing fragments.
The number of LDB1 (A) and CTCF (B) interactions is lower in mouse fetal brain
when compared to primary erythroid cells. Furthermore, the mean and the median
of
the distance between either LDB1 (C) or CTCF (D) interaction partners is lower
in
mouse fetal brain cells when compared to mouse primary erythroid cells.
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Figures 6 to 10: Visualization of interaction matrices for mouse fetal brain
(Figure 6),
mouse fetal liver (Figure 7), human HB2 (Figure 8), human TEV (Figure 9), and
human HEV (Figure 10) cells all for -2Mbp region and using in the
visualization a
logarithmic frequency range and a rainbow colour code. The pictures show
clearly the
superior resolution and quality of T2C and with a direct visual readout, that
the
genome is organised in subchromosomal domains, consisting of chromatin loops
which form loop aggregates/rosettes. This is species specific (compare Figures
6 and
7, with Figures 8-10), tissue/cell specific (Figures 6 and 7 and Figures 8-
10), depends
on the activity of genes (Figures 6, 7, 8 and 9), and the presence of
structurally
relevant proteins such as cohesin (Figures 8 and 9). Thus, the structure also
depends
to disease states in which genetic or structural changes, change the
interactions
(Figures 6 and 7, or Figures 9 and 10).
Figure 11: Simulated chromatin models description and relation/evaluation of
spatial
distances between genomic markers, in the lmmunoglobulin Heavy Chain Locus and
the Prader-Willi/Angelmann Syndrome region: a, Volume rendered images of
simulated Random-Walk/Giant-Loop and Multi-Loop-Subcompartment Models. As a
starting conformation with the form and size of a metaphase chromosome (top),
rosettes were stacked (alpha). From such a starting configuration, interphase
chromosomes in thermodynamic equilibrium, were decondensed by Monte-Carlo and
relaxing Brownian Dynamics steps. A volume rendered image of the simulated
Random-Walk/Giant-Loop model containing large loops (5 Mbp) is shown (left;
beta).
Note that the large loops do not form distinct structures but intermingle
freely (left;
beta). In contrast, in a volume rendered image of the simulated Multi-Loop-
Subcompartment Model, containing 126 kbp sized loops and linkers, the rosettes
form distinct chromatin territories in which the loops do not intermingle
freely (middle;
gamma In an image of the simulated RW/GL model containing 126 kbp loops and 63
kbp linkers, again distinct chromatin territories are formed but in contrast
to the MLS
model no subcompartments form (right; delta). B: Random-Walk Giant Loop and
Multi-Loop-Subcompartment Models: indicates the RW/GL model in which large
loops are attached to a non-DNA backbone. shows the simulated model containing
a
chromatin linker between loops. MLS model is shown containing 126 kbp loops
and
linkers with individual rosettes spanning 1-2 Mbp.
Figure 12: Simulated interaction maps for different crosslink probabilities
(di: distance
of interaction) for chromatin models description and relation/evaluation of
spatial
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distances for various Multi-Loop-Subcompartment models (model parameters: loop-
size/linker size / modelname).
Figure 13: Simulated interaction maps for different crosslink probabilities
(d,: distance
of interaction) for chromatin models description and relation/evaluation of
spatial
distances for various Random-Walk/Giant-Loop models (model parameters: loop-
size/linker size / model name).
SUMMARY OF ASPECTS OF THE INVENTION
The present inventors have developed a new technique entitled 'Targeted-
Chromatin
Capture' (T2C) in order to overcome the disadvantages of 5C and HiC.
T2C employs a selective enrichment of the 3C ligation products from one or
more
region(s) of interest in order to identify the interactions within a domain
and the
compartmentalization of one or several specific regions of the genome. The
region of
interest may be a large (e.g. many megabase-sized) continuous genomic region
or
may alternatively be a collection of smaller regions (a few megabases each).
Every captured restriction fragment can be used as a "viewpoint", identifying
the
nucleotide sequences which interact with that sequence in the three-
dimensional
genome structure. The output of T2C provides a local interaction map with
restriction
fragment-level resolution. The method involves considerably less sequence
efforts
and less intricate bioinformatics analysis than the Hi-C method. The method
also is
not hampered by the limitations of the 5C method since T2C also identifies
interactions of the fragments within the targeted region(s) with regions
outside of the
targeted region(s).
Thus, in a first aspect, the present invention provides a method for analysing
the
interaction of one or more nucleotide sequence(s) from one or more region(s)
of
interest with other nucleotides sequences in a three-dimensional DNA
structure,
comprising the steps of:
(a) providing a sample of cross-linked DNA;
(b) digesting the cross-linked DNA with a first restriction enzyme;
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(c) ligating the cross-linked nucleotide sequences;
(d) reversing the cross-linking;
(e) fragmenting the ligated molecules from (d);
(f) hybridising the fragments from (e) to one or more oligonucleotides
representing the
sequences which are adjacent to the cleavage site of the first restriction
enzyme in
order to enrich for the ends of the nucleotide sequences that have been
ligated to
another nucleotide sequence in step (c); and
(g) analysing the nucleotide sequence of the enriched fragments in order to
identify
the nucleotide sequences involved in interaction(s).
The method may be used for analysing the interaction of one or more nucleotide
sequence(s) from one or more genomic region(s) of interest with other
nucleotides
sequences in three-dimensional chromatin structure.
The first restriction enzyme may be any restriction enzyme that recognises a 6-
8 bp
recognition site.
The first restriction enzyme may be selected from the group consisting of
Bg/II,
HindIII, EcoRI, BamHI, Spel, Pstl and Ndel.
In step (e) of the method, the ligated molecules may be fragmented by
digestion with
a second restriction enzyme, such as an enzyme recognises a 4 or 5 bp
nucleotide
sequence recognition site or even a dinucleotide sequence.
The second restriction enzyme may be selected from the group consisting of
TspEl,
MaeII, Alul, Nialll, Hpall, FnuDII, Mael, Dpnl, Mbol, Haelll, Rsal, Taql,
CvRI,
Msel, Sth1321, Dpnll, Sau3A1 and Mn/I.
Alternatively, in step (e), the ligated molecules may be fragmented by
mechanical
means, such as shearing or sonication.
Alternatively the first restriction enzyme may be any restriction enzyme that
recognizes a 4-6 base pair recognition site (where the 6bp is a degenerate
sequence)
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in which case the second restriction enzyme would be replaced by a non
specific
nuclease or mechanical means of shearing. This would result in a higher number
of
oligonucleotides for hybridisation (see below) and a higher resolution of the
interactions, because there are more primary restriction fragments.
In step (f), the one or more oligonucleotide probe(s) may be spotted on a
microarray
or captured on beads, or alternatively be present in solution, which are later
captured
on beads.
The oligonucleotide probe(s) may recognise a sequence adjacent to the
restriction
site of the first restriction enzyme, such as a sequence within 100bp of the
restriction
site of the first restriction enzyme.
In step (f), the nucleotide sequence fragments may be hybridised to a set of
oligonucleotide probes which comprises a plurality of oligonucleotides, each
of which
hybridises to a sequence which is adjacent to the digestion site of the first
restriction
enzyme on a nucleotide sequence from the genomic region of interest.
The set of oligonucleotide probes comprises probes specific to substantially
all the
restriction fragments obtainable by treating the genomic region(s) of interest
with the
first restriction enzyme.
An adapter sequence may be ligated to one or both ends of the nucleotide
sequence
fragments from (e) before step (f) such that the ligated nucleotide sequence
fragments may be captured on the array by hybridisation, amplified and/or
sequenced
or allow the distinction of different samples hybridised to the same sets of
oligonucleotide probes. The adapter may contain a specific address sequence
that
allows one sample to be distinguished from another sample. All sequences with
a
particular address sequence are then known to originate from one particular
sample.
Step (g) of the method may involve high throughput sequencing of the enriched
nucleotide sequence fragments.
Step (g) may be followed by bioinformatical analysis and/or visualisation of
the
interaction(s).
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The region of interest (such as the genomic region of interest) may comprise a
genetic locus of interest.
The region of interest may be about 1-50 MB in length altogether.
The method of the present invention may be used to analyse the interaction of
a
particular genetic element with other nucleotides sequences in three-
dimensional
structure, if in step (g) only the sequence of the enriched nucleotide
sequence
fragments comprising the particular genetic element are analysed in order to
identify
the nucleotide sequence(s) involved in interaction(s) with the genetic
element.
The genetic element may comprise a binding site for a transcription factor or
an
insulator or barrier element.
The genetic element may be in the region of interest, for example an element
frequently involved in or close to a genomic region that is rearranged or
deleted in
disease.
The method of the present invention may also be used to determine the
expression
status of a gene by analysing the number, type or density of interactions in a
region of
interest which comprises the gene.
The method may be used to compare gene activity between two samples, by
analysing both samples and comparing the number, type or density of
interactions in
a region of interest.
The method may be used to identify which protein, such as a transcription
factor is
responsible for particular interactions.
The samples may, for example be: from different tissues from the same subject;
from
a single subject over different time points; from equivalent tissues from
different
subjects (e.g. healthy/diseased/suspected diseased subjects).
The method may be used to identify one or more DNA-DNA interactions that are
indicative of a particular disease state by analysing a sample of cross-linked
DNA
from a diseased and a non-diseased cell, a difference between the interaction
of
nucleotides sequences in three-dimensional chromatin structure between the DNA
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sequences from the diseased and non-diseased cells showing that the DNA-DNA
interaction or pattern of DNA-DNA interactions is indicative of a particular
disease
state.
The method of the invention may be used in the diagnosis or prognosis of a
disease
or syndrome caused by or associated with a change in a DNA-DNA interactions.
In
this respect, step (a) involves providing a sample of cross-linked DNA from a
subject;
and and step (g) involves comparing the interaction between the DNA sequences
with
that of an unaffected control; a difference between the control and the
subject being
indicative that the subject is suffering from the disease or syndrome or being
indicative that the subject will suffer from the disease or syndrome.
The disease may be an inherited genetic disease, or a somatic genetic disease
such
as cancer.
In a second aspect, the invention also provides an assay method for
identifying one
or more agents that modulate the three dimensional structure of DNA comprising
the
steps of:
(a) contacting a sample with one or more agents; and
(b) performing the method of the first aspect of the invention, wherein step
(a)
comprises providing cross-linked DNA from the sample;
wherein a difference between (i) DNA interactions in the presence of the agent
and
(ii) DNA interactions in the absence of the agent is indicative of an agent
that
modulates the three dimensional structure of DNA.
T2C offers significant advantages over known 5C or HiC methods, for example:
= every restriction fragment as opposed to 5C can serve as a 'viewpoint' and
all their interactions can be identified whether they are over short or long
distances or to other chromosomes
= the compartmentalization of the genome can be identified in the regions
of
interest without requiring the large sequence effort that was required for
HiC, thus reducing cost significantly
= a better coverage and resolution of the locus is obtained when compared
to other techniques. The resolution of the T2C is based on the restriction
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enzyme used but is often of the order of 1-10 Kb (average 4-5kb for a 6bp
recognition restriction enzyme). This provides a significantly better
resolution than the usual 40Kbp bins obtained with the usual HiC.
DETAILED DESCRIPTION
The present invention relates to a method for analysing the interaction
between
nucleotides sequences in a three-dimensional DNA structure.
THREE-DIMENSIONAL DNA STRUCTURES
The term "three dimensional DNA structure" means a structure comprising DNA
which has a higher order structure that the DNA double helix, forming, for
example,
loops and folds, similar to the higher order structure of an amino acid
sequence in a
protein molecule. The structure may be composed solely of DNA, or may comprise
in
addition other molecules, such as proteins. Chromatin is an example of a
complex
between DNA and proteins.
The method of the invention is ideally suited for analysis of the three
dimensional
chromatin architecture of genomes.
The primary functions of chromatin are 1) to package DNA into a smaller volume
to fit
in the cell, 2) to provide anchor points on the DNA to allow mitosis, and 4)
to control
gene expression, DNA replication and repair. The most abundant protein
components
of chromatin are histones that compact the DNA.
The structure of chromatin depends on several factors. The overall structure
depends
on the stage of the cell cycle: during interphase the chromatin is
structurally loose to
allow access to RNA and DNA polymerases that transcribe and replicate the DNA.
The local structure of chromatin during interphase depends on the genes
present on
the DNA: DNA coding genes that are actively transcribed are more loosely
packaged
and are found .associated with RNA polymerases (referred to as euchronnatin)
while
DNA coding inactive genes are found associated with structural proteins and
are
more tightly packaged (heterochromatin). Epigenetic chemical modification of
the
structural proteins in chromatin also alter the local chromatin structure, in
particular
chemical modifications of histone proteins by methylation and acetylation. As
the cell
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prepares to divide, i.e. enters mitosis or meiosis, the chromatin packages
more tightly
to facilitate segregation of the chromosomes during anaphase.
In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct
chromosome territories. Recently large megabase-sized local chromatin
interaction
domains have been identified, termed "topological domains" (Dixon et al (2012,
Nature 485, 376-380). These domains correlate with regions of the genome that
constrain the spread of heterochromatin. The domains are stable across
different cell
types and highly conserved across species, indicating that topological domains
are an
11:1 inherent property of mammalian genomes.
The topological domains also interact with each other suggesting a possibly
higher
order structure of the genome into a series of rosette like structures.
The method of the invention may be used to identify and characterise
topological
domains or higher order structures within a genome, chromosome or part
thereof.
The spatial organisation of the genome is intimately linked to its biological
function, so
it is important to understand higher order genomic structure.
Although the method of the invention is ideally suited for analysis of the
three
dimensional chromatin architecture of genomes, it can be applied to analyse
nucleotide sequence interaction in any three-dimensional structure.
Nucleic acids, such as DNA, can spontaneously form a "quaternary structure"
with
itself, other nucleic acids and other molecules, such as proteins. The method
of the
invention can be used to analyse the three-dimensional architecture of any
nucleic
acid-containing structure. For example the method could be used to investigate
and
verify the hierarchical assembly of artificial nucleic acid building blocks
used in DNA
nanotechnology.
REGION OF INTEREST
The present invention involves analysing the interactions between nucleotide
sequence(s) in a region of interest with other nucleotide sequences.
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The region of interest may be a genomic region of interest within one (or
more)
chromosomes.
The region of interest may comprise a particular genetic locus of interest. A
genetic
locus is the specific location of a gene or DNA sequence or position on a
chromosome. The genomic region of interest may comprise a particular locus,
such
as the sequence of a particular gene, together with one or both flanking
regions. The
region of interest may, for example, comprise the sequence of about 1, 2, 3 or
4 MB
on both sides of the gene.
The "other nucleotide sequences" i.e. the nucleotide sequences with which the
nucleotide sequences within the region of interest interact, may themselves be
located in the region of interest, or they may be from other regions, such as
other
parts of the same chromosome(s) of from a different chromosome. Interactions
with
such regions may change in case of disease when the regulation of genes has
changed or when genes are lost.
DNA
.. The 3D DNA structure may comprise genomic DNA ¨ consisting of or comprising
one
or more genomic loci.
METHOD
The method of the invention includes the following steps:
(a) providing a sample of cross-linked DNA;
(b) digesting the cross-linked DNA with a first restriction enzyme;
(c) ligating the cross-linked nucleotide sequences; and
(d) reversing the cross linking.
These first four steps of the method of the invention are analogous to those
of
Chromosome Conformation Capture (3C) which is described in Dekker et al (2002)
Science 295:1306; and 4C (Capture and Characterise Colocalised Chromatin),
which
is described in WO 2007/004057.
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A 3C-like template may be prepared using known methods, such as the method
described by Splinter et al., (2004) Methods Enzymol. 375, 493-507. Briefly, a
sample ¨ such as cells, tissues or nuclei ¨ is fixed using a cross-linking
agent ¨ such
as formaldehyde. The primary restriction enzyme digestion is then performed
such
5 that the DNA is digested in the context of the cross-linked nucleus.
Intramolecular
ligation is then performed at low DNA concentrations, which favours ligation
between
cross-linked DNA fragments (ie. intramolecular ligation) over ligation between
non-
cross-linked DNA fragments (ie. intermolecular or random ligation). Next, the
cross
links are reversed and the DNA can be purified. The 3C template that is
yielded
10 contains restriction fragments that are ligated because they were
originally close in
the nuclear space.
Since a primary restriction enzyme is used to digest the DNA prior to the
intramolecular ligation step, an enzyme recognition site for the primary
restriction
15 enzyme will separate the first (target) nucleotide sequence and the
nucleotide
sequence that has been ligated. Accordingly, the primary restriction enzyme
recognition site is located between the first (target) nucleotide sequence and
the
ligated nucleotide sequence (ie. the ligated second sequence).
CROSS-LINKING
Cross-linking agents ¨ such as formaldehyde ¨ can be used to cross link
proteins to
other neighbouring proteins and nucleic acid. Thus, two or more nucleotide
sequences can be cross-linked via proteins bound to (one of) these nucleotide
sequences. Cross-linking agents other than formaldehyde can also be used in
accordance with the present invention, including those cross-linking agents
that
directly cross link nucleotide sequences. Examples of agents that cross-link
DNA
include, but are not limited to, UV light, mitomycin C, nitrogen mustard,
melphalan,
1,3-butadiene diepoxide, cis diaminedichloroplatinum(II) and cyclophosphamide.
Suitably, the cross-linking agent will form cross-links that bridge relatively
short
distances ¨ such as about 2 A - thereby selecting intimate interactions that
can be
reversed.
Cross-linking may be performed by, for example, incubating the cells in 2%
formaldehyde at room temperature ¨ such as by incubating 1 x 107 cells in 10
ml of
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DMEM-10% FCS supplemented with 2% formaldehyde for 10 min at room
temperature.
DIGESTION WITH RESTRICTION ENZYME
The cross-linked DNA is digested with a first restriction enzyme.
Restriction endonucleases are enzymes that cleave the sugar-phosphate backbone
of DNA. In most practical settings, a given restriction enzyme cuts both
strands of
duplex DNA within a stretch of just a few bases. The substrates for
restriction
enzymes are sequences of double-stranded DNA called recognition
sites/sequences.
The length of restriction recognition sites varies, depending on the
restriction enzyme
that is used. The length of the recognition sequence dictates how frequently
the
enzyme will cut in a sequence of DNA.
Restriction enzymes which recognise a 4 bp sequence of DNA, together with
their
restriction sites, include: AATT (TspEI), ACGT (MaeII), AGCT (Alul), CATG
(N1a111),
CCGG (Hpall), CGCG (FnuD11), CTAG (Mael), GATC (Dpnl, Dpnll, Sau3A1 & Mbol),
GCGC (Hhal), GGCC (Haell1), GTAC (Rsal), TOGA (Tag!), TGCA (CviR1), TTAA
(Msel), CCCG (Sth1321), CCGC (Acil) and CCTC (MnI1)
Restriction enzymes which recognise a 6 bp sequence of DNA, together with
their
restriction sites, include: AACGTT (Ac11), AAGCTT (Hind111), AATATT (Sspl),
ACATGT (BspLU111), ACCGGT (Agel), ACGCGT (Mlul), ACTAGT (Spel), AGATCT
(Bg111), AGCGCT (Eco4711I), AGGCCT (Stul), AGTACT (Scal), ATCGAT (Clal),
ATGCAT (Avail!), ATTAAT (Vspl), CAATTG (Mfel), CACGTG (PmaCI), CAGCTG
(Pvull), CATATG (Ndel), CCATGG (Ncol), CCCGGG (Smal), CCGCGG (Sac11),
CCTAGG (AvrI1), CGATCG (Pvul), CGGCCG (Xmall1), CGTACG (Sp11), CTCGAG
(Xhol), CTGCAG (Pstl), CTTAAG (AR), GAATTC (EcoRI), GACGTC (AatI1),
GAGCTC (Sac!), GATATC (EcoRV), GCATGC (Sph1), GCCGGC (Nael), GCGCGC
(Bse131), GCTAGC (Nhel), GGATCC (BamH1), GGCGCC (Narl), GGGCCC (Apal),
GGTACC (Kpnl), GTATAC (Snal), GTCGAC (Sall), GTGCAC (ApaLI), GTTAAC
(Hpal), TACGTA (SnaBI), TCATGA (BspH1), TCCGGA (BspMII), TCGCGA (Nrul),
TCTAGA (Xbal), TGATCA (Bc11), TGCGCA (Mstl), TGGCCA (Ball), TGTACA
(Bsp14071), TTATAA (Psil), TTCGAA (Asull) and TTTAAA (Ahern).
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Restriction enzymes which recognise a 7 bp sequence of DNA, together with
their
restriction sites, include: CCTNAGG (Saul), GCTNAGC (Espl), GGTNACC BstEll and
TCCNGGA Pfol.
Restriction enzymes which recognise an 8 bp sequence of DNA, together with
their
restriction sites, include: ATTTAAAT (Swal), CCTGCAGG (Sse8387I), CGCCGGCG
(Sse232I), CGTCGACG (SgrDI), GCCCGGGC (Srfl), GCGATCGC (Sgfl),
GCGGCCGC (Notl), GGCCGGCC (Fsel), GGCGCGCC (Ascl), GTTTAAAC (Pmel)
and TTAATTAA (Pad).
There are also restriction enzymes which recognise degenerate sequences which
means that two or more bases are possible at a particular position in the
recognition
sequence effectively resulting in 3or 5bp sequences of DNA that is recognized.
One
can also use a combination of enzymes to effectively recognise 2bp, for
example the
combination of HpyCH21V, Mspl, HinPII and Taql effectively recognizes the 2 bp
sequence CG.
The first restriction enzyme (or combination of enzymes) may recognise a 2, 4,
5, 6, 7
or 8 bp sequence of DNA.
The first restriction enzyme may, in particular, be a 6-cutter, such as
HindlIl or BgIII.
The second restriction enzyme (or combination of enzymes) may recognize a 2 or
4
bp sequence of DNA or be replaced by a nonspecific nuclease (in which case
only a
limited digestion would be applied) or mechanical fragmentation.
LIGATION AND REVERSAL OF CROSS-LINKING
The digestion step is then followed by ligation under diluted conditions that
favour
intra-molecular interactions and joining of the DNA via the compatible ends.
Ligation may induced by the addition of a ligase enzyme.
The ligation reaction may be performed at a low DNA concentration, such as
about 1-
5 ng/p.I.
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Cross-linking may be reversed by the addition of an agent such as proteinase
K.
FURTHER STEPS OF THE METHOD
The method of the invention may also involve:
e) fragmenting the ligated DNA, for example with a second restriction enzyme
(such
as a 4bp recognition enzyme) or other nucleases or by mechanical shearing. In
the
latter cases the DNA ends may be repaired to become blunt-ended to allow the
addition of an adapter sequence
(f) ligating on an adapter sequence that contains a specific sequence that
allows the
distinction between samples (the other sample containing a linker with a
different
specific sequence) and/or sequences that allow hi-throughput sequencing
g) hybridise the ligated sample to one oligonucleotide probe or set(s) of
oligonucleotide probes representing one or more genomic loci. The one or
set(s) of
oligonucleotides are selected on the basis of their proximity to the first
restriction site
as in step (a) and their hybridisation temperature. The latter is dependent on
their
length and base composition. Different oligonucleotide probes in a set should
have
similar hybridisation/melting temperatures. Moreover they should be unique to
prevent the hybridisation of repetitive DNA. The oligonucleotide probes can be
attached to a solid surface or contain a tag such as biotin that allows
capture on a
solid surface preferably streptavidin beads.
(h) stringently wash the hybridised solid surface after hybridisation to
remove the non
hybridised material.
(i) elute the hybridised material
(j) sequence the hybridised material for example by paired end IIlumina
sequencing
(k) use bio-informatics to map the sequences back to the genome and generate a
matrix of interaction
FRAGMENTATION
The ligated DNA molecule may be fragmented by various methods known in the
art,
such as digestion with a second restriction enzyme or other nucleases; using
radiation or heavy ions; or mechanical means such as sonication or shearing.
The second restriction enzyme should cut DNA more frequently than the first
restriction enzyme used in step (b) of the method. The second restriction
enzyme
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may recognise a shorter or more common stretch of DNA (recognition site) than
the
first restriction enzyme.
If the first restriction enzyme is a 6-8 bp cutter, the second restriction
enzyme may be,
for example, a 2 or 4-cutter.
The second restriction enzyme may, for example, be a 4-cutter such as Dpn II
of
NIalll.
The second restriction enzyme (or combination of enzymes) may recognize a 2 or
4
bp sequence of DNA or be replaced by a nonspecific nuclease (in which case
only a
limited digestion would be applied) or mechanical fragmentation. There are a
large
number of non-sequence specific nucleases, such as Micrococcal nuclease or
DNasel.
Following mechanical methods, such as shearing, non specific nucleases or
treatment using radiation or heavy ions, the ends of the nucleotide sequences
may
need to be 'repaired' by standard methods to allow the next steps.
ADAPTER
An adapter may be ligated to the ends of the fragments from step (e) for
sequencing
purposes, i.e. to enable sequence analysis for methods such as the IIlumina
method.
The adapter may comprise an address sequence. Different address sequences are
used for different samples to allow multiplexing (hybridisation of different
samples to
the same set of oligonucleotide probes) where the address sequence allows the
matching of a sequence with the sample it was derived from. Address sequences
are
useful when multiple samples or internal spiking is used.
It is preferable for the adapter sequence to be added before hybridisation. It
is
possible to add them on by ligation after hybridisation but it is likely to be
less efficient
as the DNA comes off the hybridisation as single stranded DNA.
HYBRIDISATION
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In step (f) of the method, the nucleotide sequence fragments are hybridised to
one or
more oligonucleotide probe(s) in order to enrich for fragments which comprise
an
interacting nucleotide sequence
5
The oligonucleotide probes are attached to or can be captured on a solid
support,
such as an array or beads (see below).
The oligonucleotide probes are designed based on the sequence(s) from the
region
10 of interest, bearing in mind the position of the restriction sites of
the first restriction
enzyme.
Each oligonucleotide probe corresponds to a sequence located close to the
first
restriction site. The ligated DNA molecule made in step (d) of the method of
the
15 invention comprises different nucleotide sequences, joined at the
restriction site of the
first restriction enzyme. The different nucleotide sequences were
"interacting" (i.e. in
close enough proximity to be cross-linked) in the three dimensional structure.
When
the ligated molecule is fragmented, some fragments will be derived from a
single
nucleotide sequence, from internal fragmentation (e.g. internal digestion by
the
20 second restriction enzyme). Other fragments will be derived from both
the interacting
nucleotide sequences.
By selecting fragments which have a sequence which is located close to the
first
restriction site, the fragments are enriched for those which represent an
"interacting
fragment" i.e. comprise a portion of two nucleotide sequences joined at the at
the
restriction site of the first restriction enzyme by the ligation step (c).
OLIGONUCLEOTIDE PROBES
Suitably, the oligonucleotide probes will be at least 15, 20, 25, 30 or 40
nucleotides in
length.
The oligonucleotide probes are designed to be as close as possible to the
restriction
enzyme recognition site of the first restriction enzyme. The term "adjacent
to" means
that the oligonucleotide probes are designed such that they recognise a site
within
about 100 nucleotides - such as about 90, 80, 70, 60, 50, 40, 30, 20, 10, 9,
8, 7, 6, 5,
4, 3, 2 or 1 nucleotide(s) away from the first restriction enzyme recognition
site.
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If the region of interest has X recognition sites of the first restriction
enzyme (RE1),
digestion with RE1 will produce X+1 fragments. These fragments will have an
RE1
recognition site at both ends, so it is necessary to design 2X oligonucleotide
probes to
encompass all fragments in the region of interest.
The library of oligonucleotide probes may comprise oligonucleotides specific
to
substantially all the restriction fragments obtained by treating the region(s)
of interest
with the first restriction enzyme. "Substantially all" in this context, means
at least 60,
70, 80, 90, 95 or 99% of the restriction fragment-flanking sites.
Occasionally it is not possible to design an oligonucleotide probe
representing one of
the ends, for example:
(I) the sequence may be repetitive
(ii) the second recognition enzyme site (RE2) may be too close to the RE1
site
(iii) there is no RE2 site between two RE1 sites. (When non-specific
nuclease or
mechanical fragmentation is used, this does not apply).
If any of the above limitations apply, oligonucleotide probes to that
particular RE1
restriction fragment or end thereof may be omitted from the set of
oligonucleotide
probes, but the oligonucleotide set would still contain oligonucleotide probes
to
"substantially all" of the RE1-flanking sites.
ANALYSIS
Once the fragments have been enriched for those representing an "interaction",
the
nucleotide sequences involved in the interaction may be characterised by
sequencing.
Pair-end sequencing may be carried our using known techniques, such as the
IIlumina system.
An adapter sequence may be ligated to one or both ends of the nucleotide
sequence
fragments from (e) preferably before or less preferred after step (f) such
that the
ligated nucleotide sequence fragments may be captured on an array, amplified
and/or
sequenced. The adapter sequence may provide an address to recognize a sample
when several samples are analysed on the same array, i.e. multiplexing. It is
possible
22
to multiplex 8 samples in one lane of an IIlumina machine presently yielding
150
million sequence reads per lane.
In more detail, the fragments may be end repaired and A-tailed, and the
indexed
adapters ligated to the A-tailed DNA fragments.
The resulting adapter-modified DNA library may be captured, eluted and PCR
amplified. In the method of the invention the fragments may not be PCR
amplified
prior to the enrichment step (step (f)).
Cluster generation and high-throughput sequencing may then be performed by
known
techniques (e.g. using the IIlumina cluster reagents and a HiSeqTm 2000
sequencer).
The interaction frequencies may be visualised by producing a two dimensional
heat
map as previously described (Liberman-Aiden et al (Science 2009 326:289-293;
Dixon et al (2012, as above). Interaction frequencies between any two loci can
be
visualised by identifying the point off the axis where diagonals originating
from each
locus intersect, in a manner similar to a linkage disequilibrium plot.
Each point on the map represents an interaction point between two fragments
(two
fragments in close proximity). The intensity of each interaction point on the
map is
relative to the frequency of interaction/proximity of the fragments which it
represents.
The points on the diagonal represent self-ligation effect as well as ligation
to the
immediately neighbouring fragments. The visualisation is basically a matrix
analysis.
SAM PLE
A sample may be any physical entity comprising DNA that is or is capable of
being
cross-linked. The sample may be or may be derived from biological material.
The sample may be or may be derived from one or more cells, one or more
nuclei, or
one or more tissue samples. The entities may be or may be derivable from any
entities in which DNA ¨ such as chromatin - is present. The sample may be or
may
be derived from one or more isolated cells or one or more isolated tissue
samples, or
one or more isolated nuclei.
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The sample may be or may be derived from living cells and/or dead cells and/or
nuclear lysates and/or isolated chromatin.
The sample may be or may be derived from cells of diseased and/or non-diseased
subjects.
The sample may be or may be derived from a subject that is suspected to be
suffering from a disease.
The sample may be or may be derived from a subject that is to be tested for
the
likelihood that they will suffer from a disease in the future.
The sample may be or may be derived from viable or non-viable patient
material.
A standard sample may be added to each experimental sample (spiking) to allow
better comparison between different sample as the samples may be normalised
using
the sequence reads of the spiking sample. The spiking sample may be from a
different species than the experimental sample to allow spiking in the form of
cells at
the first step, alternatively the spiking sample may have its own address or
be from a
.. different species when spiking at later stages in the procedure.
ARRAY
Typically, the set of oligonucleotide probes will be immobilised on a support
or be
captured on a solid support such as beads. Supports (eq. solid supports) can
be
made of a variety of materials - such as glass, silica, plastic, nylon or
nitrocellulose.
When attached to a solid support it is preferably rigid and have a planar
surface.
Supports typically have from about 1-10,000,000 discrete spatially addressable
regions, or cells. Supports having about 10-1,000,000 or about 100-100,000 or
about
1000-100,000 cells are common. The density of cells is typically at least
about 1000,
10,000, 100,000 or 1,000,000 cells within a square centimeter. In some
supports, all
cells are occupied by pooled mixtures of oligonucleotide probes or a set of
oligonucleotide probes. In other supports, some cells are occupied by pooled
mixtures of probes or a set of oligonucleotide probes, and other cells are
occupied, at
least to the degree of purity obtainable by synthesis methods, by a single
type of
oligonucleotide.
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For a restriction enzyme recognising a >6 bp recognition sequence, a single
array of
about 2 x 750,000 oligonucleotide probes can be used to cover, for example,
the
complete human or mouse genome, with 1 oligonucleotide probe at each side of
each
restriction site.
OLIGONUCLEOTIDE PROBES IN SOLUTION
Oligonucleotide probes in solution may contain a moiety that can be captured
on a
solid surface, such as oligonucleotides containing a biotin that can be
captured by
streptavidin beads. Hybridisation in solution may be more efficient.
Capture may take place after hybridisation
HYBRIDISATION
The term "hybridisation" as used herein shall include "the process by which a
strand
of nucleic acid joins with a complementary strand through base pairing".
Nucleotide sequences capable of selective hybridisation will be generally be
at least
75%, 85%, 90%, 95% or 98% homologous to the corresponding complementary
nucleotide sequence over the length of the oligonucleotide probe. Selectivity
is
determined by the salt and temperature conditions during the hybridisation.
"Specific hybridisation" refers to the binding, duplexing, or hybridising of a
molecule
only to a particular nucleotide sequence under stringent conditions (e.g. 65 C
and
0.1xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na-citrate pH 7.0}). Stringent
conditions
are conditions under which a oligonucleotide probe will hybridise to its
target
sequence, but to no other sequences. Stringent conditions are sequence-
dependent
and are different in different circumstances. Longer sequences hybridise
specifically
at higher temperatures. Generally, very stringent conditions are selected to
be about
5 C lower than the thermal melting point (Tm) for the specific sequence at a
defined
ionic strength and pH. The hybridisation temperature is the temperature below
the
melting temperature (Tm) and the closer the hybridisation temperature is to
the Tm
the more stringent the hybridisation is, meaning that mismatched DNA sequences
will
not hybridise to each other. The oligonucleotide sequences should be in excess
over
the genomic DNA to ensure efficient, preferably complete and thereby
quatitative
hybridisation. Typically, stringent conditions include a salt concentration of
at least
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about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3.
Stringent
conditions can also be achieved with the addition of destabilising agents -
such as
formamide or tetraalkyl ammonium salts.
5
The invention will now be further described by way of Examples, which are
meant to
serve to assist one of ordinary skill in the art in carrying out the invention
and are not
intended in any way to limit the scope of the invention.
10 EXAMPLES
Example 1 - T2C identifies known long-range interactions
To test the method and to compare it with other methods, the inventors first
chose the
IGF/H19 region on human chromosome 11 that has previously been used to study
15 the role of cohesion and CTCF for chromosomal long-range interactions
and for
which Hi-C and 4C data are already available for comparison (Figure 2).
A set of array-based oligonucleotides were designed mapping near the ends of
all the
BOII fragments covering an approximately 2.1Mbp region of the H19 locus,
totaling
20 524 oligonucleotides corresponding to 344 BglIl fragments. A number of
BglIl
fragments did not allow the design of an oligonucleotide representing one of
the ends
because the sequence was either repetitive or the 4bp recognition enzyme site
(N1a111) was too close to the BglIl site or completely absent from the Bg111
fragment.
The crosslinked Bg111 restricted DNA was ligated, decrosslinked, digested with
NIalll
25 enzyme and hybridized to the oligonucleotide array after decrosslinking
(see
Methods).
Analysis of the sequenced ligation products first with a 40 kb binning of the
genome
as used for HiC demonstrated that T2C reveals a similar overall interaction
pattern as
observed by Dixon et at ((2012), as above) for IMR90 cells (interactions
outside the
area or with other chromosomes are also observed but not shown). This is also
consistent with the previously observed conservation of overall architectural
features
like topological domains between different cell lines (Figure 2A+B).
However, with T2C, an interaction map at restriction fragment resolution
(Figure 2C)
was obtained, revealing a lot more detail with respect to the general
chromatin
organization of the region and contacts between genes and their regulatory
elements.
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To compare this chromatin structure information of T2C the data were compared
with
4C data and the 4C data obtained for a particular CTCF viewpoint were plotted
next
to the interaction data observed for the same viewpoint present in the T2C
data
(Figure 2D).
Although there are some variations in the read coverage of the individual
interactions,
the same interactions can be observed by 4C and T2C. The T2C method therefore
yields reproducible results, faithfully detects the fragments that interact
(or are in
close proximity), clearly reproduces the overall genomic structure in
topological
domains and gives resolution around the 4-5kbp expected for a 6bp recognition
restriction fragment.
Example 2 - T2C identifies different interaction networks based on different
biological materials
In order to also test whether different gene expression states can be detected
in
different biological tissues with different chromatin interactions, T2C was
applied in in
vivo mouse primary erythroid cells from mouse fetal liver and brain cells from
E12.5
mice. The well-studied p-globin locus was used as an example in a region of -2
MB
around the gene. It is well established that as P-globin is expressed more
highly in
primary erythroid cells compared to fetal brain cells, a denser number of
interactions
is expected around the gene and between the gene and its locus control region
(LCR)
in this cell type. The p-globin region was digested with HindlIl as the 6bp
enzyme
and 799 oligonucleotide probes were designed to cover the ends of the HindlIl
fragments in the locus (724 fragments, many of which are repetitive) and after
crosslinking re-digested with Dpnl I.
The analysis of the hybridised fragment after cleavage with Dpnll showed 5
topological domains in the region of interest (-2 MB) in both mouse primary
erythroid
cells and mouse fetal brain cells with many interactions within each
topological
domain. The topological domains also interact with each other suggesting a
possibly
higher order stucture of the genome into a series of rosette like structures.
Although
the number of topological domains between the different biological materials
appears
to be the same the interactions within and between the topological domains
appear to
be less dense in mouse fetal brain cells comparing mouse primary erythroid
cells
(Figure 3). Zooming in on the all the P-globin region shows all the well-known
interactions in the p-globin locus in the fetal liver material. The
interactions such as
between the p-globin promoter and LCR and between the LCR-3'HS1 are clearly
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visualised (Figure 3). These are absent from the fetal brain sample. Moreover,
it is
possible to identify new additional interactions further away than the ones
reported
until now for the p-globin promoter. These are located as far as ¨1Mbp from
the 13-
globin promoter.
The interactions of the binding sites of an important regulatory transcription
factor in
fetal liver cells, the LDB1 complex or the structural factor CTCF, was also
compared.
LDB1 is highly enriched on the P-globin locus and its LCR in mouse primary
erythroid
cells when compared to fetal brain cells. By visualizing only the restriction
fragments
containing the LDB1 or CTCF transcription factor binding sites as determined
by
ChIP-seq (e.g. Soler et al (2010) Genes Dev;24(3):277-89), it is possible to
immediately deduce which interactions out of all the interactions involve the
LDB1
complex or CTCF (Figure 4). It is also clear that in mouse primary erythroid
cells,
more LDB1 occupied restriction fragments have interactions with other
positions in
the locus when compared to mouse brain cells (Figure 4). In addition, the mean
of
the distance between two fragments in close proximity is larger in fetal liver
cells
suggesting this area of the genome is less condensed in the fetal liver when
compared to fetal brain (Figure 5).
T2C is therefore a useful tool to detect topological domains and the different
interactions within domains depending on the expression status of the genes
such as
= the active f3-globin locus in primary fetal liver cells versus the same
silent locus in
fetal brain. In addition, the high level of resolution of the interaction
allows novel
observations such as shown for the p-globin locus LDB1 binding sites and size
of
loops. Deletions within such a locus as for example in p-thalassemia caused by
DNA
deletions would be immediately visible through the change of interaction
signals.
Discussion
The importance of the role of chromatin interactions in the regulation of the
genes is
well established. However, there is an increasing need of a quick, easy and
affordable techniques to provide the information about the interactions and
the
compartmentalization of the genome. T2C satisfies these needs. Every
restriction
fragment can serve as a 'viewpoint' and all their interactions, either sort or
long or to
other chromosomes (not shown here), can be identified. Thus, multiple 3C-seq,
4C or
Sc experiments do not have to be performed. Moreover,
with T2C, the
compartmentalization of the genome can be identified in the regions of
interest
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without requiring the large sequence effort that was required for HiC, which
increases
the costs significantly.
Due to the design of T2C, a better coverage and resolution of the locus is
obtained
when compared to other techniques. The resolution of the T2C is based on the
restriction enzyme used. Digesting crosslinked chromatin from primary
erythroid cells
and HB2 cells with HindlIl or BglIl resulted on an average resolution of 2.9Kb
and
6.1Kb respectively. This provides a significantly better resolution than the
usual
40Kbp bins obtained with HiC. Moreover by adding the appropriate addresses in
the
oligonucleotides ligated on to the fragments (after the second cleavage before
hybridisation) for sequencing purposes allows the multiplexing of different
samples to
the same set of oligonucleotides as the address sequence identifies the sample
from
which it was derived. Multiplexing further reduces the cost of T2C.
Furthermore, comparing, T2C with 3C-seq and HiC for the Igf2 locus and with
previously published 3C-qPCR data for the 8-globin locus, the same topological
domains and interaction networks are identified. All these reveal the
strengths of T2C
as a tool to identify all the interactions and the compartmentalization of a
specific
regions of the genome.
Thus T2C is an affordable, cost effective tool to explore the local spatial
organization
of the genome and chromatin interactions without requiring laborious
procedures or
massive sequencing efforts.
Materials and methods for Examples 1 and 2
Chromatin isolation and library preparation
Nuclei from mouse primary erythroid cells from mouse fetal liver E12.5, mouse
fetal
brain cells and a human breast endothel cell line (HB2) were isolated and
crosslinked.
The chromatin was digested with a 6-cutter (HindlIl for mouse cells and BglIl
for the
HB2 cells), ligated and de-cross-linked. From the resulting libraries 50pg DNA
was
digested with a frequent 4-cutter (DpnII or NIalll for the mouse cells, NIalll
for the HB2
cells). All these steps were performed according to the 3C-seq protocol
previously
described (Stadhouders, R. et al. Nat Protoc 8, 509-524 (2013)).
A microarray for the 8-globin locus was designed containing unique
oligonucleotides
as close as possible to the HindlIl restriction sites spanning ¨2 MB around
the gene
(chr7: 109875617-111971734, mm9). For the Igf2 locus, unique oligonucleotides
29
were designed close to BglIl restriction sites (ch11: 1091427- 3228670, hg19)
spanning an area of ¨2.1 MB. The ligation products enriched by hybridization
on the
microarray were sequenced by paired-end sequencing yielding more than 100
million
unique read pairs for the first and the second design respectively.
The final library is prepared for analysis on the IIlumina Cluster Station and
HiSeq
2000 Sequencer according to the IIlumina TruSeqTm DNA protocol with
modifications.
In short, 20 pg of the digested library was purified using AMPure XP beads
(Beckman Coulter) and end-repaired. The now blunt-ended fragments were A-
tailed
using the Klenow exo enzyme in the presence of ATP and purified again using
AMPure XP beads. Indexed adapters (IIlumina) were ligated to the A-tailed DNA
fragments with subsequent purification using AMPure XP beads.
Array capturing
The resulting adapter-modified DNA library was hybridized for 64 hours at 42 C
on a
custom-made NimbleGen Tm Sequence Capture 2.1M capture array according to the
NimbleGen Sequence Capture array protocol on the NimbleGen Hybridization
System. The captured DNA fragments are eluted from the hybridised array and
purified using MinElute columns (Qiagen). The captured DNA fragments are
amplified by PCR using Phusion polymerase as follow: 30 s at 98 C, 24 cycles
of (10
s at 98 C, 30 s at 60 C, 30 s at 72 C), 5 min at 72 C final extension. PCR
products
are purified using AMPure XP beads and eluted in 30 pl of re-suspension
buffer. One
microliter is loaded on an Agilent Technologies 2100 Bioanalyzer using a DNA
1000
assay to determine the library concentration and to check for quality.
Cluster generation and high throughput sequencing
Cluster generation is performed according to the IIlumina Cluster Reagents
preparation protocol. Briefly, 1 pl of a 10 nM TruSeq DNA library stock is
denatured
with NaOH, diluted to 9-10 pM and hybridized onto the flowcell. The hybridized
fragments are sequentially amplified, linearized and end-blocked according to
the
IIlumina Paired-end Sequencing user guide protocol. After hybridization of the
sequencing primer, sequencing-by-synthesis is performed using the HiSeq 2000
sequencer with a 101 cycle protocol according to manufacturer's protocol. The
sequenced fragments were denaturated with NaOH using the HiSeq 2000 and the
index-primer was hybridized onto the fragments. The index was sequenced with a
7-
cycle protocol. The fragments are denaturated with NaOH,
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sequentially amplified, linearized and end-blocked. After hybridization of the
sequencing primer, sequencing-by-synthesis of the third read is performed
using the
HiSeq 2000 sequencer with a 101-cycle protocol.
5
Example 3 - Determination of the 3D structure of genomes:
The dynamic three-dimensional chromatin architecture of genomes and the
obvious
co-evolutionary connection to its function - the storage and expression of
genetic
information - is still, after -130 years of concentrated research, one of the
central
10 issues of our time. In this example the detailed 3D architecture of the
mouse and
human genome can be determined directly for the first time from a few to the
mega
base -pair level by already visual means combining a novel superior selective
high-
throughput high-resolution chromosomal interaction capture of all physical
genomic
interactions (HRHTICIC2), scaling analysis, and polymer simulations: the
clearly existing
15 and differently compacted chromatin fibre is folded into loops of -30-
150 kbp which
form defined loop aggregates/rosettes (sub-chromosomal domains) of -500-1500
kbp
connected by a linker. Complex (helical) loop and loop-loop architectures
exist and
interactions vary only to a minor but significant extent between different
cell types or
functional states. Beyond, scaling analysis proves shows the tight
evolutionary
20 entanglement between DNA sequence and genome architecture. Consequently,
this
finally opens the path to detailed architectural "sequencing" of genomes and
thus true
systems genomics at the limit of the "genomic uncertainty principle", all of
which is of
fundamental importance for genome understanding and R&D of diagnosis and
treatment.
Despite the fact that the structure and function of genomes obviously co-
evolved as
an inseparable system to allow the physical storage and expression of genetic
information, neither the dynamic three-dimensional higher-order architecture
of
genomes, its spatial and temporal modifications, nor its relation to
functional multi-
dimensional interaction and regulatory networks have yet been determined in
detail
since the discovery of the cell nucleus by A. van Leeuwenhoek in the 17th
century
and many another more recent landmark result: the discovery/description of
metphase chromosomes by C. W. Nagli (1842)/W. Hofmeister (1848), the DNA by
Miescher (1869), the DNA double helix by R. E. Franklin, L. C. Pauling, J. D.
Watson,
and F. H. Crick, (1953), the nucleosome by R. Kornberg (1973)/A. Olins & D.
Olins
(1974), and the 3D structure of the nucleosome by K. Luger (1997), up to
sequencing
of the entire human genome at the turn of the millennium. Beyond, it became
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apparent genome organization and function indeed build a systems genomic
(Knoch,
2003) entity responsible for gene expression and thus for the intrinsic
differences
between individuals and their disease history as well as the receiver of
functional
environmental genome alterations and thus eventually external disease causes.
The size, structure, and complexity of genomes span scales from 10-9 to 10-5 m
and
10-15 to 105 s, and thus result in huge experimental challenges: Already how
nucleosomes are spaced, positioned, remodelled, and whether/how nucleosome
chains fold into fibers at physiological salt concentrations are a matters of
continuing
debate: e.g. Finch and Klug (1976) proposed a regular solenoid, in vivo
neutron
II) scattering experiments revealed a fiber diameter of 30 5nm as a
dominant nuclear
feature, in recent contrast to no compaction at all, or to highly polymorphic
and
dynamic function dependent structures without which nucleosome concentration
distributions, dynamic and functional properties as diffusion of
macromolecules, and
the scaling of the DNA sequence are unexplainable.
The higher-order chromatin architecture has been a matter of even greater
debate for
more than a century: Light microscopic studies by Rabl (1885) and Boveri
(1909) led
to hierarchical self-similar models, suggesting a territorial organization,
before
electron microscopy suggested a more random interphase organization - as in
the
models of Comings (1968, 1978) and Vogel & Schroeder (1974). In the radial-
loop-
scaffold model of Paulson & Laemmli (1980) chromatin loops attached to a
nuclear
matrix/scaffold should explain the condensation degree of metaphase
chromosomes.
According to Pienta & Coffey (1977, pub1ished1984), these loops persisted in
interphase, and formed stacked rosettes in metaphase. Micro-irradiation by C.
Cremer & T. Cremer (1974,1982) had already and fluorescence in situ
hybridization
(FISH) by C. Cremer & T. Cremer (2001), P. Lichter (1988) and publications
thereafter finally confirmed a territorial organization of chromosomes, their
arms, and
of sub-chromosomal domains during interphase including their structural
persistence
during metaphase (de-)condensation (the -850 G, Q, R, and C ideogram bands
split
in -2500 sub-chromosomal domains). Whereas, chromatin rosettes were visualized
by electron microscopy but not taken seriously in the western hemisphere
(Erenpreisa, 1989, Belmont & Bruce (1994) proposed also based on electron
microcopy the helical hierarchy chromonema fiber (CF) model, for the intra-
(sub-
territorial folding. Around the same time, spatial distance measurements
between
small FISH labeled genetic regions, led due to architecturAL "demolition" to
the
Random-Walk/Giant-Loop (RW/GL) model with the first analytical looped polymer
description by Sachs (1995; Yokota, 1995; Yokota, 1997; Knoch, 1998; Knoch,
2002),
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in which 1 to 5 Mbp loops are attached to a non-protein backbone., Thereafter,
a
combination of distances measurements using structure preserving FISH
technology,
high-resolution microscopy, and huge parallel polymer simulations of
chromosomes
and entire cell nuclei, only could result in the rosette Multi-Loop-
Subcompartment
(MLS) model in which 60 to 120 kbp loops form rosettes connected by a similar
linker.
Again in vivo measurements of the nucleosome concentration distributions, and
the
dynamic and functional properties as diffusion of macromolecules are only
compatible
with a small loop aggregate/rosette like chromatin folding and the scaling of
the DNA
sequence also predicts this since otherwise the patterns found here are
to unexplainable otherwise.
Beyond, it be-Came apparent, since physical interactions are at the heart of
functional
chemical reaction and thus process chains, that short regulatory elements
containing
several binding sites for transcription factors regulate gene transcription
often via
huge genomic separations, and thus the resulting changes in their (physical)
interaction likelihood, are responsible for changes in gene expression, since
either the
preformed architecture or the modification or new formation of such
structures, e.g.
loops, is associated with spatial proximity, and thus changed interaction
probability. It
seems also obvious already by logical reasoning, that in the formation of
these
structures factors of the transcription cascades seem to play major role
directly or as
a dual or multiple use case as e.g. CTCF or cohesion. Consequently, both it
has
become apparent that genome architecture and functional regulation are
responsible
systems genomically via the transcription cascade for are not only responsible
for the
intrinsic differences between individuals and their disease history, but are
in turn also
the receiver of functional environmental genome alterations and thus
eventually
external disease cause.
To determine whether i) a locally more or less compacted chromatin fiber, ii)
folded in
loop aggregates/rosette exists (being consistent with all these experiments,
and every
a functional requirement in respect to the genomic "live" cycle from a few to
the mega
base pair level), iii) whether there is a general scaling behavieour of this
architecture,
iv) in agreement with long-range correlations of DNS sequence itself, and
whether
this is in agreement with v) novel in vivo measurements, a novel selective
high-
resolution high-throughput chromosomal interaction capture of all physical
genomic
interactions (everything with everything) approach was developed - HRHTiCIC2,
which
also opens the path to efficient and cheap architectural sequencing of genomes
for
diagnosis and treatment - in essence (see Sup. Method): i) starting with -107
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cultured/prepared cells, ii) the cells are formaldehyde fixed (i.e. DNA-DNA,
RNA-
RNA, DNA-RNA, protein-protein, DNA-protein, RNA-protein and more complex
genomic crosslinks are formed), iii) permeablized to allow intra-nuclear
restriction with
a first restriction enzyme, iv) large dilution by extraction of the
crosslinked fragments
to allow re-ligation primarily within these complexes, before v) de-
crosslinking,
purification, and final shortening of the DNA chimeric fragments to sizes
<500bp by a
second high-frequent restricting enzyme or by sonication (for highest
resolution).
Then, vi) a cleaned regional HRHTiCIC2 DNA interaction fragment library is
produced
using DNA capture arrays (bead capturing is also possible) with -109-1019
molecules
per unique and hybridization optimized oligo (i.e. the capture is always in
the linear
regime and far from saturation), which sequentially are directly placed next
to the first
-restriction enzyme. vii) After high-throughput sequencing, the obtained
sequences are
trimmed to contain only a sequence piece up to the first restriction enzyme,
then
mapped first to the whole reference genome and in case of using two
restriction
enzymes also against a masked sequence containing only the regions between 1st
and 2nd restriction enzyme, to use finally only 100% uniquely mapped
sequences.
This novel selective HRHTICIC2 approach has great advantages: 0 the limiting
factors
compared to other interaction capture techniques now are only the sequencing
capability/costs and the chosen relation between resolution of the first
restriction
enzyme, size of the captured region, interaction frequency range, and number
of
multiplexed experiments: e.g. a -500 bp fragment resolution, in a 2 Mbp
region, with
a 1-106 interaction frequency range, and 10-100fold multiplexing can be easily
achieved sequencing 10-100 lanes (note: also several regions can reside on one
capture array). ii) Due to the design of the oligo position, the maximum of
data
cleanness and thus the maximum interaction information with the minimum
sequencing is reached. iii) Beyond, the entire process has been optimized for
structure preservation (see Sup. Methods), which is the point also during high-
resolution FISH, where already slight differences have lead historically to
different
chromosome models. This includes also the minimization of distortions and DNA
loss
in each step - often achieved by a delicate/subtle laboratory/bench handling.
Notably,
no known structure distortion, (cost driving primers), or PCR steps are
involved until
sequencing here.
Beyond, with possible fragment length of down to 50-100 bp (persistence length
of
free DNA on average -50nm or -140 bp; typical protein/nucleosome binding
region
-200-500 bp) the fundamental limits of this method are not only reached, but
also
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more importantly what is introduced here as the genomic uncertainty principle
originating in the individuality of each high-resoluted interaction with a
unique
individual probabilistic fragment setting/condition/surrounding in each cell
at a given
moment in time, which is destroyed by the measurement - hence the classic
definition of an uncertainty principle: i) already the cell population has a
distribution of
cell statuses and functional differences, ii) each fragment has a more or less
dynamic
(and thus stable or variable) individual DNA, RNA, protein, restriction
association,
thus the entire crosslinking, restriction, and re-ligation has a different
individual
efficiency, and, of course, iii) the DNA sequence in relation to the oligo
hybridization
capture, the sequencing, and mapping add also to this. Thus, in the end only
probabilistic analysis and statements can be drawn as known from quantum
mesoscopic systems in general and well known from the classic light double-
slit
experiment. Currently, there are also no means for any sensible correction,
since at
least currently the actual state of the influencing factors/parameters are
innumerous,
.. incalculable (especially due to their non-linearity), different for every
single fragment,
and besides that destroyed by the measurement. This has always been the case
for
any interaction capturing kind, although effects were averaged out by the low
resolution (allowing for nevertheless senseless but in their effect not
harmful
corrections), but now the fundamental limit is reached. This opens the
opportunity to
perceive the interaction information integrated over all these effects in its
completeness and beauty at this fundamental level with unprecedented insights.
To investigate the chromatin fiber conformation and the 3D architecture with
the
necessary resolution and biological impact, the human chromosome 11p 15.5-
15.4,
i.e. the IGF/H19 region, and the mouse chromosome 7qE3-F1, i.e. the p-globin
region
were selected, since both -2.1 Mbp regions are classic well studied by FISH
and 3C
examples of epigenetic and local control region regulation. By using Bgl II
and Hind III
as first and Nalll as second restriction enzyme this results in many fragments
down
to -200-500 bp with an average of 6121 and 2915 bp, respectively. To study at
even
.. higher resolution tThe chromatin fiber conformation was then analysed at
high
resolution in general we also investigated roughly (and with low sequencing
coverage) 15 regions of in total 99.5 Mbp on 10 different mouse chromosomes
with
-50 to 500 bp andto an average fragment size of 549 bp. Thus, we reach
molecular
and nucleosomal (average nucleosomal repeat length -200 bp, thus 3-6 kbp
correspond to -15-30 nucleosomes on average) and even subnucleosomal
resolution, and thusi.e. the level of the genomic uncertainty principle. To
investigate
differences between species, cell lines, and functional/architectural
differences the
35
human HB2 cell line and the cohesin cleavable TEV/HRV RAD21-eGFP cell line
system, and mouse fetal brain (p-globin inactive) and fetal liver (3-globin
active) cells
were used. To investigate the chromatin fibre formation also fetal liver cells
were
used. With ¨107 input cells concerning sequencing, the corresponding material
(e.g.
the two different states) were multiplexed on the capture array to guarantee
equal
conditions. One or two lanes were sequenced either in the same sequencing run
or
different ones. Due to the various effects only sequences unique in the entire
genome
with a reasonable mismatch rate (to account for sequencing differences/errors
to and
in the reference genome) and cleaned for sequences only mapping between the
first
and second restriction sites.
Thus, sSorting and plotting the regional interactions in an upright squared
interaction
matrix (with two mirrored triangle halves) with a logarithmic and rainbow
coloured
frequency range shows directly the validity of the experiments itself and the
unprecedented frequency range distribution spanning 6 orders of magnitude in
general and 4-5 excluding the diagonal. Thus, also rare interactions with a
frequency
of 104 to 10-5 can be visualized in this setting of region size, fragment
resolution, and
sequencing effort. This could easily be increased by 2-4 orders of magnitude
changing this relation. Beyond, the relation of the average cumulated entries
per
.. fragment in relation to the ¨107 million input cells shows an estimated
¨0.1-1.0%
efficiency of HRH-riCIC2. Beyond, tThe patterns show clearly that a level is
reached
where the uncertainty principle in the statistical limit reached the stable
probabilistic
level, since images from different sequencing lanes for the same experiment
whether
multiplexed or not show only minor statistical deviations.
Determination of the 3D structure by visual means from T2C
All the interaction matrices of different experiments are reproducible
reproducibly
more or less empty, i.e. there is no prominent uniform noise or background,
despite
the high number of sequence reads and despite most diagonal elements showing
entries of non- or self ligated fragments and thus demonstrating that a
capturing oligo
was present and worked. The "emptiness" is also clearly structured and not
arbitrary
and appears the same in replicates to an extremely high degree of detail, i.e.
neither
are interactions suddenly appearing statistically nor are clustered
statistically
.. somewhere near more prominent interactions. Thus, taking into account that
information from definitely >104 cells survives the procedure, noise could in
principle
appear at any step of the procedure, and even assuming an unlikely highly
biased
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distortion of a normal distributed noise signal towards e.g. interactions, the
signal to
noise ratio must be >105-106.
Even more surprising already visually the interactions themselves are even
more
.. striking in respect to the appearance of clearly distinct patterns on all
scales of
genomic separation and even the fact that patterns consistently are or are not
reemerging on other scales (which they have to do because genomes are scale
bridging systems) and also show immediately that the whole T2C procedure
actually
works despite of the numerous and nonlinear a parameter involved, since the
chance
that such patterns arise is unthinkably small. A first comparison to known
viewpoints
reveals agreement of T2C with e.g. 3Cseq although with much cleaner and
sharper
interactions for the same fragment distribution since with T2C no PCR
broadening of
interactions appears. Consequently, the detailed interactions patterns can be
now
interpreted even more easily.
Determination of the conformation of the chromatin fibre by visual inspection:
On the smallest genomic scale, there is clearly a denser interaction pattern
in the
band parallel to the diagonal for genomic separations < 5-10 kbp (i.e. < 25-50
.. nucleosomes), compared to bigger genomic separations. This pattern varies
independent of the local fragment resolution (which nevertheless needs to be
considered) and consists of distinct interactions with noninteracting "gaps"
in
between, in contrast to a homogenous e.g. Gaussian like interaction "smear"
decreasing for increased genomic separation. Thus, consequently visual
inspection
directly shows already, that on this scale ¨ the scale of DNA/nucleosomes ¨
stable
and defined interactions exist and thus since these interactions are the
outcome of
spatial proximity indicate that a compaction of nucleosomes exist into an
irregular but
nevertheless locally defined structure, i.e. the notion of a fiber applies,
which one
could in general terms due its variation call "quasi-fiber" with an average
density etc..
Obviously, a structurally everywhere exactly identical and uniform fiber as
proposed
by the helical chromatin fiber model would lead in contrast to a homogenous
band-
like subpattern, and a constantly dynamic random walk of nucleosomes would
also
result in a homogenous interaction pattern with a Reighley distributed
interaction
decrease as function of genomic separation. Thus, by visual inspection one can
read-
out directly the existence of a chromatin "quasi-fiber", its local interaction
and thus
compactness structure naturally averaged over the entire T2C procedural
boundaries.
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Determination of the subchromosomal structural domains by visual inspection:
On the biggest scale the appearance of square-like domains in the range of
several
hundred to -1-1.5 Mbp, with a sharp border and interacting with other domains
is also
immediately visible (although more prominently in the human compared to the
mouse
case) with several striking general properties: First the interaction
frequency within
the domains for the moment neglecting their substructure has in general an
average
uniform height and drops on the edge to another uniform height defining the
interaction between subdomains. Thus, there is a staircase like behaviour of
interactions in contrast to the often thought general continuous interaction
decrease
with growing genomic separation and a clear and defined interactions with
other
domains. Second at the borders of the domains there is a clear transition or
linker
region between domains although the interactions between domains is especially
strong and complicated since near the diagonal also the chromatin quasi-fibre
comes
into play and since the linker is in structural terms is very flexible.
Consequently,
these results proof again the existence of structurally stable sub-chromosomal
units,
which are relatively stable and interact with each other especially well at
their borders
as described in the historic overview. Beyond, already on this level, the on
average
uniform interaction within the domain and the sharp drop at the edge very
clearly
indicates already towards a loop-aggregate and even rosette like structure of
the
domains connected by a linker, since one big loop, a random walk or fractal
globule
like folding all would not lead to the sharp edge and defined behaviour found
here.
Determination of the conformation of the chromatin higher-order structure,
i.e. the
loop/aggregate/rosette folding of chromosomes by visual inspection.
On medium scale, and thus the sub-chromosomal domain level, the interaction
pattern is characterized by again clearly distinct gaps between the
interactions, which
are arranged in a crossed linear or grid-like pattern. Interestingly the
linear pattern
continuous outside the sub-chromosomal domain and "crosses" there with the
linear
pattern originating from the subsequent sub-chromosomal domain. Beyond, the
general lower general interaction frequency level outside the domains and the
less
complicated interaction pattern seen there, allows to follow the linear
pattern back into
the domains revealing that the much simpler/clear patter outside clearly also
is
.. present within the domains, but there enriched and more complicated by
additional
interactions. One now can follow such a line back to the diagonal and take
this as
starting viewpoint following vertically to the next relevant interaction
(which one can
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follow horizontally from outside into the domain again). Then one localizes
horizontally again the interaction point on the diagonal. Repeating this gives
a second
interaction point on the diagonal and now one can prove that in most cases
this
second interaction also interacts with the first and thus starting point.
Thus, by hand a
grid of interactions can be constructed. This can be enhanced by projecting
the
interactions vertically and horizontally, resulting in a peak-like pattern
along the
chromosome sequence, whose peaks coincide with the crossed linear pattern.
This is
more prominent in the human compared to the mouse case. Since interactions on
scales on tens of kilo base pairs are and can only be considered especially as
chromatin looping, this means that several consecutive loops whose loop bases
are
visualized by the interactions have coinciding loop bases, i.e. a loop
aggregate with a
core, and thus also a rosette of loops with a more or less clear core. The
gaps
between interactions and the grid-like pattern also show that no other folding
like
random-walk giant loops, a chromonema like, or fractal globule pattern cannot
be the
origin of that since they all would result in a homogenous interaction
pattern, without a
clear domain border and clearly not a distinct domain border. This would also
be the
case for a non-compaction into a quasi-chromatin fibre, which a sea-of
nucleosome
organization would predict, resulting in huge and very dynamic interaction
possibility.
Notably, the data structure on different scales also proof also the assumption
that on
all scales interactions can indeed be cross-linked and depend on different
underlying
cross-linkable agents is correct, and thus that crosslinks between a specific
DNA
location or protein etc. create such patterns is very unlikely. Additionally
the simple
pattern is more complex due to the variation in the compaction density of
chromatin
and the fact that within the loops additional interactions take place of
various forms:
on a large scale simple loop or even super-helical like patterns seem likely,
whereas
on a low scale the patterns around the major loop base interactions indicate
the
structure of the rosette core and the local chromatin compaction and the
entanglement of both there. Although the experiments at highest resolution was
made
to investigate the chromatin fibre conformation in more detail in general and
thus
involved less deep sequencing, an overall assessment of these data indeed
resulted
in finding several such structures which could be attributed to loop
aggregates/rosettes and a detailed core structure with in and outgoing loops
which
leads to special interaction patterns. The interactions between the domain and
their
pattern can be attributed thus to two origins: On the one hand the loop
aggregate/rosette cores of subsequent domains can due to the relatively low
number
of loops and thus density and loop dynamics interact very easily. On the other
hand in
a cell population there are also mitotic chromosomes where the condensation
degree
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is naturally very high. Thus, the pattern explains both consistently the
organization
and its dynamics through the cell cycle and again this is only possible with a
compacted chromatin fibre since otherwise, core interactions between
aggregate/rosette cores would be shielded by polymer fibre exclusion.
Determination of the 3D structure by visual inspection as a function of
different cell
types, or treatment of the cells, or diseased state:
To investigate the architectural change in general as function of specie, cell
type,
regional, functional or structural differences due to regulation or deliberate
system
distortion the human IGF/H19 11p 15.5-15.4 region was investigated, and the
mouse
13-globin 7qE3-F1, in human HB2 and the TEV/HEV cells, and mouse fetal brain
(FB)
and liver (FL) cells: Whereas the general general domains are clearly the same
in
HB2, TEV, HEV, and FB and FL cells, and thus in different cell types of the
same
specie, but different between human and mouse at least due to the region
chosen.
On a finer degree of detail human HB2 cells seem to show a more and denser
interaction pattern within the domains in comparison to the TEV/HEV system.
Comparing FB to FL cells does not show such an obvious difference: actually
the
differences a very subtle often belonging to single or a small group of
interactions as
can be shown for the P-globin locus where an additional loop is formed as
predicted
from earlier experiments. Nevertheless, the term small here is relative, since
such a
single loop formation actually activates the I3-globin transcription, i.e. an
entire
pathway and thus the entire property of a cell can be changed. Beyond,
cleaving
cohesion (said to play a major constitutive role in genome architecture) in
the
TEV/HEV system leads, however, also not to dramatic changes, well actually
only
slightly more and more evenly spread interactions suggesting a slightly higher
flexible/dynamic architecture but not as big as previously thought as in a
genome
wide analysis. Thus, cohesion cannot be single and definitely not major/singe
component responsible agent, but instead is one of obviously several
components
influencing/forming genomic architectures and shows the evolutionary balance
between flexibility and stability of genome architecture. Consequently, these
variations show not only the reproducible quality of T2C and its analysis
under
different conditions and that a clear general genome architecture exists, but
also that
a the reached level of the genomic uncertainty principle in essence locally
every detail
is a variation of the great theme to be considered to fine-regulate the
system. Thus,
obviously in only ¨2 Mbp big genomic regions a wealth of interaction data is
present
to be carefully analysed in detail.
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Determination of 3D chromatin higher order structure by Monte-Carlo and
Brownian-
Dynamics simulations in comparison to T2C
To explore and understand this behaviour on all scales independently and in a
clearer
5 manner with preset conditions recently polymer models have been developed
to
evaluate (not to fit) in general experimental results, designs and hypotheses
about the
three-dimensional genome organization. There the resolution is based on
stretchable,
bendable polymer segments, and volume exclusion with a resolution comparable
to
-1-2.5 kbp and featuring the Random-Walk/Giant-Loop model in which large loops
10 (0.5-5.0 Mbp) are linked by a linker resembling a flexible backbone, and
featuring the
Multi-Loop Subcompartmerit (MLS) model with rosette-like aggregates (0.5-2
Mbp)
with smaller loops (60-250 kbp) connected by linkers of variable sizes (60-250
kbp).
These simulations were enhanced and for the first time two-dimensional spatial
distance and interactions maps (for different crosslink probabilities and
extent) were
15 calculated with extremely high statistical validity. Visual comparison
reveals
immediately, that all above described effects interpretations are in agreement
with the
simulations, and beyond the interactions are a function of all model
parameters even
in slight details considering that no nucleosomes where modelled here: i) in
general
the interaction degree depends on the interaction and crosslink probability,
ii) the
20 domain size, domain separation, and spacing of loops are proportional to
their size,
iii) the interactions between the domains depend on the linker size, the size
and
number of loops, i.e. density of the rosettes. Thus, the subtle combination of
density
of rosettes due to loop size, loop number, chromatin fibre persistence and the
thus
resulting exclusion effects leading eventually for high numbers to spread out
and
25 shielding effects of rosettes, as well as the subtle influence on the
interaction pattern
between entire domains. The linker between domains and its proportionality to
inter-
domain interactions is as clearly visible as well as non equilibration effects
which we
deliberately show here to create an understanding of the interactions of loops
at
aggregate/rosette borders and similar effects. Also the in general large
emptiness of
30 interaction matrices and the link to the existence of a dedicated
chromatin fibre is
obvious and also proves that the crosslink probability, radius, and frequency
can be
estimated to be relatively low although since the relation contains a too
complex
parameter set not unambiguously fittable. The models show also clearly the
special
behaviour at the loops bases of the rosettes, which in reality due to the
variation of
35 the compaction in reality might be more complex, although experiments
with highest
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41
resolution show, that various such structures exist but have to be
investigated in
much further detail in the future.
Determination of 3D chromatin higher-order structure by determination of the
scaling
behaviour of T2C from the scaling behaviour of the frequency of interactions
as a
function of the genetic distance between interactions and in comparison to the
scaling
behaviour of Monte-Carlo and Brownian-Dynamics simulations and the scaling of
the
long-range correlations in the DNA itself:
to To investigate in a unified scale-bridging manner the behaviour from
scales of several
base pairs, via the mega base pair and subdomain level, up to the scale of
entire
chromosomes and thus nucleus (spanning scales from 10-9 to i05 m), we
introduced
scaling analysis and showed its capabilities: The scaling of the interaction
frequency
as function of the genomic separation the different simulated models given by
shows
clearly long-range scaling, with a multi-scaling behaviour with a fine-
structure
attributable to i) the general interaction decrease, i.e. spatial distance
increase, ii) the
sub-chromosomal domain like structure, iii) the aggregated loop/rosette like
structure
in the subdomains. All parameter variations can be re-found in a changed
scaling
behaviour on scales here. This is in agreement, with other measures of
scaling. Thus,
there is no uniform scaling as e.g. seen in self-similar fractals bridging and
the same
on all scales, but its deviation shows the substructure in domains and loops.
The scaling behaviour of the different regions is in comparison a scaling
behaviour of
subset of a chromosome and thus is dominated by the local architecture
deviations
from the general scaling behaviour and is also compromised by the amount of
interactions used here. Nevertheless, the scaling behaviour shows i) a long-
range
multi-scaling behaviour with a fine-structure, ii) with subtle but not in
general
differences for the various specie, cell type, functional/distortion
differences.
.. The scaling behaviour of interaction frequencies as function of their
genomic
separation of different published experiments on the entire genome shows also
i) a
long-range multi-scaling behaviour with a fine-structure, ii) with subtle but
not in
general differences for the various specie, cell type, functional/distortion
differences.
Both experimental and modelled scaling behaviours, however, only agree with
loop
aggregated/rosette like genome architectures with loop aggregates (0.5-2 Mbp)
with
smaller loops (60-250 kbp) connected by linkers of variable sizes (60-250
kbp).
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Since what is near in physical space should also be near in DNA sequence
space,
since mutations of all sorts will be biased by genome architecture itself, we
also
investigated the correlation behaviour of the DNA sequence by the most simple
correlation analysis possible, i.e. the mean square deviation of the base pair
composition within windows of different size for two different human and mouse
entirely sequenced strains: i) long-range power-law correlations were found
using
correlation analysis on almost the entire observable scale, ii) with the local
correlation
coefficients showing a species specific multi-scaling behaviour with close to
random
correlations on the scale of a few base pairs, a first maximum from 40 to 3400
bp,
to and a second maximum from 105 to 3x105 bp, and iii) an additional fine-
structure is
present in the first and second maximum. The behaviour in general and in
detail is
stronger in the human compared to the mouse case, but within the different
chromosomes nearly identical and only deviating for certain chromosomes to a
somewhat larger extent. The behaviour on all scales is not only equivalent to
the
.. long-range multi-scaling of the genome architecture in detail but also at
the right
scales. Thus, the second maximum found corresponds to in size and position to
the
sub-chromosomal domains. Especially on the fine structural level, the
previously
already proven association to nucleosomal binding at the first general maximum
can
now be extended to second maximum as well and associated with the looped
structure in there as previously predicted.
Beyond, the interaction scaling at highest resolution shows the same behaviour
across different chromosomes as the scaling behaviour of the DNA sequence in
the
same maximum: although a fine-structure cannot be found due to the
experimental
resolution, the general behaviour with a broad peak and a stronger interaction
decrease on scales above ¨4 kbp strongly suggests, in both the interaction
experiment and in the DNA sequence, that a compacted chromatin fibre indeed
exists.
Methods for Example 3 with a detailed description of T2C:
HB2 Cell Line and Cell Culture
HB2 cells (1-7HB2, a clonal derivative of the human mammary luminal epithelial
cell
line MTSV1-7) were cultured in DMEM supplemented with 0.2 mM L-glutamine, 100
units/ml penicillin, 100 mg/m1 streptomycin, 10% FCS, 5 pg/ml
hydroxycortisone, and
10 pg/ml human insulin. In a previous 3C study we confirmed the karyotype and
the
DNA methylation of several regions.
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TEV/HRV Cell Line System and Cell Culture
The cleavable TEV/HRV RAD21-eGFP cell line system is a HEK293T cell line
system, which was transfected with a pRTS-1 vector encoding for a cleavable
RAD21-eGFP fusion protein and an siRNA for endogenous RAD21 knock-down. Both
are expressed by doxycycline induced activation of a bidirectional promoter
inbetween and thus simultaneously. For the RAD2-eGFP fusion-protein, a
cleavable
RAD21, where the first RAD21-separase cleavage site is replaced by that of the
3C
protease of the human rhinovirus (HRV protease) using a PCR-based mutagenesis
(the second cleavage site remained unchanged to ensure less cell cytotoxicity)
was
inserted before eGFP. The tobacco etch virus protease (TEV protease), does not
recognize the HRV cleavage site and thus can act as a control. The endogenous
RAD21 knock-down sequence allows knock-down with the following 3'UTR-directed
siRNA's:
5'-ACUCAGACUUCAGUGUAUA-3' (Scc1-1),
5'-AGGACAGACUGAUGGGAAA-3' (Scc1-2).
For generation of the TEV/HRV RAD21-eGFP cell line system the original HEK293T
cell line was cultured in DMEM supplemented with 0.2mM L-glutamine, 100
units/ml
penicillin, 100 mg/ml streptomycin, 10% FCS, and was grown at 37 C and 5% CO2.
For the transfection Lipofectamine 2000 (Invitrogen) according to the
instructions of
the manufacturer was used. Cells carrying the vector were selected by growth
in 150
pg/mL hygromycin containing medium. Single clones were picked and analysed for
expression of RAD21cv and RAD21wt constructs and depletion of the endogenous
RAD21 three days after induction with 2 pg/ml doxycycline. The resulting
TEV/HRV
RAD21-eGFP cell line was as well cultured in DMEM supplemented with 0.2mM L-
glutamine at 37 C and 5% CO2.
For experiments and to activate transgene expression with HRV (or TEV which
serves as a control, thus a transfection takes place, but no cleavage) the
cells were
cultured for 3 days in the presence of 2 pg/ml of doxycycline. Thereafter,
cells were
split, reseeded until 50% confluency and transfected with HRV or TEV vectors
using
Lipofectamine 2000 (Invitrogen) again according to the instructions of the
manufacturer. 24 hours after protease transfection the cells were used for the
experiment.
Cell Preparation from Mice
For mouse fetal liver and fetal brain cells, -10 embryos on day 12.5 of
pregnancy
from one to two transgenic FVB/N mice were used for the -10 million cells
required
44
by the experiment to have a complex enough cell population and enough DNA in
the
end to be sequenced: The mice were cleaned with 70% Et0H and the abdomen was
opened to remove the cervix containing the embryos, before cutting them lose
and
removing them from the yolk sac and placenta. Small and underdeveloped embryos
were discarded. The embryos are collected in petri dishes on ice with 0.5 ml
10%
FCS/PBS. Then the fetal liver and brain were dissected from the embryos and
collected in tubes (1 ml) on ice containing again 500 p110% FCS/PBS. The cells
were
then resuspended with a P1000/1m1 plastic pipette tip and connective tissue
was
digested by adding 25 pl of a 2.5% Collagenase stock (0.125% end
concentration)
and incubated for ¨ 45 min at 37 C. Thereafter, the cell suspension was
transferred
to falcon tubes with 12 ml 10% FCS/PBS at room temperature and thereafter was
gently squeezed through a scraper mesh which was placed inside a 6-well plate
using
again a P1000/1 m1 plastic pipette tip. The mesh was washed with 2m1 10%
FCS/PBS
at room temperature to get all the cells from the mesh. The resulting single
cell
suspension was again collected in falcon tubes with an end volume of 12 ml
10%FCS/PBS at room temperature. Notably, we tried to keep the stress of the
cells to
the minimum, to avoid any damage to the cell nuclei. Both after the
resuspension, the
Collagenase treatment, and/or after the scraping of fetal liver and brain
material/cells
were spotted on glass slides to check for cellular and especially nuclear
integrity by
microscopy, with or without staining of the nucleus with DAPI (or eventually
any other
immunofluorescence or fluorescence in situ hybridization).
HRHTiC/C2 Crosslinking/Fixation of Cells
For crosslinking/fixation of the genome and the entire cells, the cells were
first
counted and their concentration adjusted to 10 million in 12 ml 10% FCS/PBS at
room
temperature and put into 15m1 polypropylene tubes (used for cell culture and
thus not
excessively absorbing/fixing cells to the tube wall, Greiner Bio One). Then
650 pl of a
37 % formaldehyde PBS solution were added, i.e. a final concentration of 1.9 %
of
formaldehyde is used for crosslinking/fixation, at room temperature for 10 min
while
softly tumbling to avoid cell aggregation. Note: the concentration of
formaldehyde for
crosslinking/fixation at this stage is ideal for the following steps and in
respect to
cell/nuclear integrity for the human and mouse cells we used here; although
this
might hold in general, cases are known where other concentrations and
incubation
times achieve better results. Thus, the tube was put on ice (from now on we
kept
everything on ice up to the 1st restriction of the DNA (see below) to avoid
any
damage of the material) and 1.6 ml of cold 1M Glycine in PBS were added to
stop the
crosslink/fixation reaction. Thereafter, the cells were spun down for 8 min at
1300 rpm
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45
at 4 C, the resulting pellet was washed in ice-cold PBS, and taken up first in
1 ml
before adding up to 14 ml of PBS, followed again by spinning down for 8 min at
1300
rpm at 4 C. After discarding the supernatant, the pellet could now also be
frozen for
storage, although we advise to straight away continue with lysis and the 1st
restriction. Again cells were spotted on glass slides to check for cellular
and
especially nuclear integrity by microscopy, with/without staining of the
nucleus with
DAPI (Note: again the cells could now also be used eventually for any other
immunofluorescence or fluorescence in situ hybridization experiment).
Preparation of Cell Nuclei and 1st Nuclear Genomic DNA Restriction
For lysis of the cells and to prepare cell nuclei, we prepared always 5 ml of
a fresh
(for full activity) lysis buffer on ice (I) consisting of 10 mM Tris pH 8.0
(50 pl 1M), 10
mM NaCI (10 pl 5M), 0,2% NP-40 (100 p110%), 100 pl 50x complete prot. Inhib.
(50X
= 1 tablet in 1 ml PBS), and filled up with up to 5 ml MilliQ (4.74 ml). The
pellet
prepared in the last step of crosslinking/fixation was taken up in 1 ml of
this lysis
buffer, resuspended and filled up with another 4 ml to a total of 5 ml and
incubated for
10 min on ice. The now free cell nuclei were spun down for 5 min at 1800 rpm
at 4 C,
the pellet was taken up in 0.5 ml of ice cold PBS safe-lock tube, and spun for
1 min at
2600 rpm a at 4 C. Again here it is possible after removal of the supernatant
and
snap-freezing to store the nuclei at -80 C. For a check we always spotted
nuclei on
glass slides to check for nuclear integrity by microscopy and/or staining of
the nucleus
with DAPI.
For the 1st restriction the nuclei were now resuspended in 0.5 ml/tubes with
1.2x
restriction buffer (60 pl restriction buffer, 440 pl MilliQ and adjusted for
BSA if
necessary) and transferred to a 1.5 ml safe-lock tube. Then to gently
permeabilize the
nuclear lamina the tubes were put to 37 C and 7.5 pl of 20% SDS (0.3%
endconcentration) were added, and incubated at 37 C for 1 h, while shaking at
900
rpm. After adding 50 pl of Triton-X-100 Tm (2% endconcentration) for further
gentle
permeabilzing the nuclear lamina, the nuclei were again incubated at 37 C for
1 h,
while shaking at 900 rpm. Note: both the SDS and Triton-X-100 step need to be
carried out with great care to avoid any decrosslinking ¨ again we checked
that by
checking the nuclei microscopically with and/or without DAPI staining. For
future
controls of the undigested material (the so called 1st unrestricted control)
now a 5 pl
aliquot was taken and stored at -20 C. Then 400 Units of the selected
restriction
enzyme was added and incubated over night (-20 h) at 37 C. For the human cells
in
all cases the restriction enzyme BglIl (Roche) was used. For the mouse cells
we used
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46
either HindlIl (Roche) or Apol (New England Biolabs) was used. Note: even
though its
optimal temperature is 50 C for Apol 37 C should be used to prevent partial
decrosslinking of the sample ;-). And again for future controls of the
restriction now a
pl aliquot was taken and stored at -20 C (the so called 1st restricted
control). After
5 the 1st restriction 40 pl of 20% SDS (enconcentration 1.6%) was added to
the
remaining sample to stop the restriction and for further breakdown of the
nuclear
lamina by incubation at 65 C for 20-25 min, while being shaken at 900 rpm.
Dilution, Re-ligation and De-Crosslinking of Restricted Genomic DNA
Thereafter, the fully digested nuclear material was diluted by transferal to a
50 ml
falcon tube and addition of 6.125 ml 1.15x ligation buffer (6.125m1: 5421 ml
MilliQ +
704 pl ligation buffer). Then 375 pl of 20% Triton-X-100 (endconcentration
1.0%) was
added and incubated in a 37 C water bath for 1 h, while being shaken every 10
min
by hand. Then 20 pl Ligase HC 5U/p1 (100 U in total, Roche) was added and
incubated at 16 C over night (-20 h) followed by an additional 30 min of
incubation at
room temperature. To de-crosslink the non-ligated and ligated DNA 30 p110
mg/ml
Proteinase K was added and incubated at 65 C in a water bath over night (-20
h).
Again for future controls of the relegation and de-crosslinking now a 5 pl
aliquot was
taken and stored at -20 C (so called re-ligation/de-crosslink control).
DNA Purification and 2nd (Re-ligated-)DNA Restriction/Sonication
For further treatment of the sample, first the DNA was purified by adding 30
p110
mg/ml RNAse (300 pg in total) and incubation for at 37 C for 30-45 min,
followed by
brief cooling to room temperature and addition of 7 ml phenol-chloroform and
.. vigorous shaking. Then the sample was centrifuged at 4,000 rmp (2200xg) for
15 min,
before the upper phase was put in a new 50 ml tube and 7 ml of MilliQ was
added as
well as 1 pl of glycogen per ml, 1.5 ml of 2M Sodium Acetate pH 5.6, and add
35 ml
of 100% ethanol to enhance the purification, gently but thoroughly mixed and
thereafter put at -80 C for 1.5-3 h. This was followed by direct
centrifugation at 4,000
rmp (2200xg) for 15 min, supernatant removal, addition of 10 ml of 70% Et0H,
resuspension, and again centrifugation at 4,000 rmp (2200xg) at 4 C for 15
min. After
supernatant removal, the pellet was dried for 20 min and was dissoluted in 150
pl of
10 mM Tris pH 7.5 at 37 C for 30 min. Again for future controls of the
relegation and
de-crosslinking now a 5 pl aliquot was taken and stored at -20 C (so called
1st
purification control).
Date Recue/Date Received 2021-05-25
47
Thereafter, the resulting re-ligated and de-crosslinked purified material was
shortened
by a 2nd restriction: First, to control the amount of DNA at this stage an
aliquot of 1 pl
was run alongside a reference sample of species-matched genomic DNA of known
concentration on a 2% agarose gel. Then the DNA was adjusted in 0.5 ml/tubes
to a
100 ng/pl concentration and restricted with the 2nd restriction enzyme by
adding 1 U
per pg of DNA of the selected restriction enzyme and incubated over night (-20
h) at
37 C. For the human cells in all cases the restriction enzyme Nlall (New
England
Biolabs) was used. For the mouse cells we used either if HindlIl was used as
1st
restriction enzyme Dpnll (New England Biolabs) or if Apol was used as 1st
restriction
enzyme sonication with 10 cycles of 15 sec on and 45 sec off.
Treatment of the Various DNA Controls
For controls of the integrity of the DNA at the different stages the following
controls
were used: i) 1st unrestricted control, ii) 1st restricted control, iii) re-
ligation/de-
crosslink control, iv) 1st purification control, and v) 2nd restriction/final
purification
control. These samples were controlled on a 2% agarose gel with corresponding
plasmid DNA, which was restricted alongside, re-ligated and purified as
external
restriction control. For controls i)-iii) the aliquots were incubated with 10
pl Proteinase
K (10 mg/ml) in 90 p 110 mM Tris pH 7.5 at 65 C for at least 1 h. The DNA was
purified by adding 3 p110 mg/ml RNAse and incubation for at 37 C for 30-45
min,
followed by brief cooling to room temperature and addition of MilliQ up to 500
pl
(-400 ml) as well as 500 pl phenol-chloroform and vigorous shaking. Then the
controls were centrifuged at 13,200 rmp for 15 min, 2 pl of glycogen per ml,
50 pl of
2M Sodium Acetate pH 5.6, and add 850 pl of 100% Et0H were added, gently but
thoroughly mixed and snap-frozen before direct procession to centrifugation at
13,200
rmp for 20 min, followed by supernatant removal, addition of 1 ml 70 Et0H,
centrifugation at 13,200 rpm at 4 C, renewed supernatant removal, pellet
drying for
20 min and dissolution in 20 pl of 10 mM Tris pH 7.5 at 37 C for 30 min.
7-2c General DNA Whole Genome Sequencing Library Preparation
In general the DNA T2C fragment library was prepared for sequencing analysis
on
the IIlumina Cluster Station and HiSeq 2000 Sequencer according to the
IIlumina
TruSeq DNA protocol with enhancing modifications from us,TruSeq DNA sample
prep
LS protocol; part#15026489 Rev. C): i) purification of the DNA fragments, ii)
end-
repair to reach blunt end status, iii) 3'-end Adenylation to avoid chimera, in
the iv)
sequencing adapter ligation including eventual multiplexing
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step, and finally v) purification of the T2C whole genome sequencing DNA
fragment
library.
Therefore, first the concentration of the T2C DNA fragment library was
measured
again for fine tuning using 1 pl of material using Quant-it dsDNA broad range
assay
kit. Then the samples were split into 4 sets of 5 pg each of the T2C DNA
fragment
library and the following complete procedure done for each of these 4 sets of
material:
i) To purify the T2C DNA library after the 2nd restriction AMPure XP beads
(Beckman
Coulter) were used by adding 1.8 pl AMPure XP beads per 1.0 pl of digested
DNA.
This was incubated at room temperature for 5 minutes, placed on the magnetic
stand
and incubated at room temperature for 5 minutes, and the supernatant was
discarded
without disturbing the beads. The beads were washed 2 times with freshly
prepared
70% ethanol, placed at 37 C for 5 minutes to let the beads dry. Then the beads
were
resuspended in 50 pl PCR grade water and incubated at room temperature for 5
minutes, placed on the magnetic stand for 5 minutes, and finally 50 pl
supernatant
was transferred to a new tube. One microliter was finally loaded on an Agilent
Technologies 2100 Bioanalyzer using a DNA 1000 assay to determine the quality
of
the purified digested DNA.
ii) For end-repair of the T2C library DNA fragments, since they were
restricted or
sonicated before with overhanging ends, 4 material sets were each in 50 pl
transferred to a 96 well plate. Since no in-line control reagent to avoid
contamination
of the material was used, 10 pl of resuspension buffer were added, followed by
40 pl
of end repair mix, and mixed thoroughly but gently pipetting the entire volume
up and
down 10 times. Then the plate was covered with a micro-seal 'B' adhesive seal
and
placed on the pre-heated thermal cycler at 30 C for 30 min. After removing the
adhesive seal from the plate, first the AMPure XP beads were vortexed until
they
were well dispersed, and 160 pl (consisting of 136 pl of AMPure XP beads mixed
with
24 pl of PCR grade water) were added to the wells and the entire volume was
again
pipetted thoroughly but gently up and down 10 times. After 15 min of
incubation, the
plate was put on the magnetic stand at room temperature for another 15 min
until the
liquid appeared clear. Then twice 127.5 pl of the supernatant was removed, and
thereafter 200 pl of freshly prepared 80 % Et0H was filled into the well of
the plate
without disturbing the beads, incubated at room temperature for 30 sec and
discarded
again without disturbing the beads. This was repeated twice before drying of
the plate
for 15 min. Only thereafter the plate was removed from the magnetic stand and
the
pellets resuspended with 17.5 pl of resuspension buffer, followed by 10 times
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thorough but gentle mixing by pipetting 10 times up and down. After incubation
at
room temperature for 2 min, the plate was put back on the magnetic stand at
room
temperature for 5 min again until the liquid appeared clear, and then 15 pl of
the clear
supernatant was removed containing the end-repaired material ready for the
Adenylation of the 3'-ends in the next step.
iii) For 3'-end Adenylation of the end-repaired HRHTiC/C DNA fragment
libraries, i.e. to
prevent the blunt ends from ligating to one another, and thus to ensure a low
rate of
chimera (concatenated template) formation during the adapter ligation reaction
in step
iv), Klenow exo enzyme in the presence of ATP was used. A corresponding single
'T'
nucleotide on the 3' end of the adapter provided a complementary overhang for
ligating the adapter to the fragment.
Therefore, 15 pl of the end-repaired T2C DNA fragment library were transferred
to a
new 0.3 ml PCR plate. Since the in-line control reagent to avoid contamination
of the
material was again not used 2.5 pl of the resuspension buffer was added,
followed by
12.5 pl of thawed A-tailing mix, pipetted thoroughly but gently up and down 10
times.
Then the plate was sealed with a microseal 'B' adhesive seal, and the plate
was
placed on a pre-heated thermal cycler at 37 C for 30 min. Immediately after
removal
of the plate from the thermal cycler, the adapter ligation took place.
iv) To ligate the sequencing adaptors using the IIlumina provided indexed
adapters #6
and #12, DNA adapter tubes and stop ligation buffer tubes were used, and
centrifuged to 600 xg , for 5 seconds. Immediately before use, the ligation
mix
containing tube was removed from the -25 C storage as recommended by IIlumina.
Since the in-line control reagent to avoid contamination of the material was
again not
used 2.5 pl of the resuspension buffer was added to the wells of another PCR
plate,
and 2.5 pl of the ligation mix was added as well. Then 2.5 pl from the
appropriate
adaptor tubes was added and thoroughly but gently pipetted up and down 10
times.
Then the plate was sealed again with a microseal 'B' adhesive seal and the
plate
centrifuged to 280xg for 1 min. Thereafter, the plate was incubated on a pre-
heated
thermal cycler at 30 C for 10 min, the plate was taken down from the cycler,
the
adhesive seal removed, 5 pl of the stop ligation buffer was added, and
thoroughly but
gently pipetted up and down 10 times.
v) To purify the sequencer adapted T2C DNA fragment libraries again AMPure XP
beads were used. Therefore, AMPure XP Beads were centrifuged until they were
well
dispersed and 42.5 pl of mixed AMPure XP Beads were added to the wells and
thoroughly but gently pipetted up and down 10 times, before incubation at room
temperature for 15 min. Then the plate was placed on the magnetic stand at
room
temperature for minimum 5 min or longer until the liquid appeared clear. Then
80 pl of
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the supernatant were removed from each well of the plate and while the plate
remained on the magnetic stand, 200 pl of freshly prepared 80% Et0H were added
without disturbing the beads, and incubated at room temperature for 30 sec.
The
complete supernatant was then removed. This Et0H wash was done twice, before
5 the still on the magnetic stand resting plate was air-dried at room
temperature for 15
min. After removal from the magnetic stand, the dried pellet was resuspended
using
52.5 pl of resuspension buffer, and thoroughly but gently pipetted up and down
10
times. After incubation for 2 min, the plate was put back to the magnetic
stand at
room temperature for minimum 5 min or longer until the liquid appeared clear.
Then
10, 50 pl of the clear supernatant was transferred to a new 0.3. PCR plate
for a second
cleanup, and 50 pl of vortexed AMPure XP beads added, and thoroughly but
gently
pipetted up and down 10 times. Then the plate was again incubated at room
temperature for 15 min, the plate was placed again on the magnetic stand at
room
temperature for minimum 5 min or longer until the liquid appeared clear. 95 pl
of the
15 supernatant were removed, while the plate still remained on the magnetic
stand, 200
pl of freshly prepared 80% Et0H was added to each well without disturbing the
beads, incubated at room temperature for 30 sec. The complete supernatant was
then removed. This Et0H wash was done again twice, before the still on the
magnetic
stand resting plate was air-dried at room temperature for 15 min. After
removal from
20 the magnetic stand, the dried pellet was resuspended using 22.5 pl of
resuspension
buffer, and thoroughly but gently pipetted up and down 10 times. Again after
incubation for 2 min the plate was put back to the magnetic stand at room
temperature for minimum 5 min or longer until the liquid appeared clear.
Finally 20 pl
of the clear supernatant from each well of the plate were collected and the
material
25 from each of the 4 in parallel treated T2C DNA fragment libraries splits
pooled.
Regional DNA Sequencing Capture Microarray Design
To achieve a high-resolution and allow for high-throughput multiplexed
sequencing
and thus to achieve a highly relevant local interaction mapping, i.e. to
achieve a high
30 quality T2C2, special capture arrays were designed to select
specifically for genome
regions of interest avoiding sequencing of unnecessary background, i.e. to
create a
regional DNA sequencing library optimized for selection of the re-ligated DNA
pieces
after the 1st restriction, i.e. directly for interactions only in specific and
relatively small
genomic regions. Therefore, in a close cooperation with NimbleGen, we designed
35 DNA oligos for 2.1 M capture microarrays, i.e. capture microarrays
capable of in
principle fishing 2.1 million different genomic sequences with the same amount
of
different oligos. To achieve a real high quality result T2C only (!) one oligo
was placed
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51
up- and one downstream as near as possible to the 1st restriction site used in
the
nuclear whole genome restriction, since the interest lies in sequencing just
each side
after re-ligation of this 1st restriction. The oligos were designed by
NimbleGen and us
for the selected regions of the human and mouse genomes using genome builts
mm9
.. and HG19 with oligo length of 72 3 bp, unique appearance (no mismatch
allowed) in
the entire genome, and with respect to best and similar, i.e. similar
hybridization,
capturing on a microarray. Then the oligos were further selected: in the case
of using
a 2nd restriction enzyme to shorten the re-ligated DNA library for sequencing
the
oligo had to be situated between the 1st and 2nd restriction site. In the case
of using
sonication to shorten the re-ligated DNA only oligos within 150 bp of the 1st
restriction
site were chosen. If only one oligo was present, which crossed either the 1st
or 2nd or
even both restriction sites, only cuts at the oligo beginning or end of in
total not more
than 10 % were allowed, i.e. that the oligos could definitely capture DNA
pieces with a
minimum of 62 bp to guaranty specificity and similar hybridization efficiency.
The
same condition was applied in the sonication case, for the 1st restriction
side.
Thereafter, we mapped the oligos on the genome and controlled by hand whether
the
conditions were fulfilled and whether the oligos were properly placed in
respect to
other genome features. For production of the microarray the number of the 2.1
million
possible different oligos nak7av was divided by the number of selected oligos
Osoiecm.i
_______________________________________________________ and then each selected
oligo spotted ilfspEredtimes, with Pispbwd ), on the
actual capturing array during the production process of the capturing
microarray by
NimbleGen. Thus, with the number of oligos used for capturing (see below)
using a
1st and 2nd restriction enzyme we can be sure to have -1010 oligo molecules
for
each different oligo on the microarray, and thus with the 107 cells we use as
input, we
are far away from saturation of the array by a factor of > 105 to 106. In the
case of the
experiment using sonication with -250 times more oligos and in total -50 times
more
genomic regions covered that is still > 102, if considering the losses in the
experimental procedures up to the capture array.
Concerning the experiments using a 1st and 2nd restriction enzyme, the balance
between the region size chosen, the resulting size of the interaction matrix,
i.e. the all
possible interactions between all restriction fragments within this region,
and the
sequencing capabilities to achieve a high frequency range of a minimum of 4 to
5
orders of magnitude for each possible interaction (we assume an average of 2
to 3
orders of magnitude, which results in a spread of 4 to 5 orders of magnitude)
was
calculated. Thus, for a sequencing capability in two sequencing lanes of -300
and
500 million sequences, i.e. 300 and 500 million sequencings of possible
interaction
52
events, with the aim of achieving on average of 100 to 1,000 sequencing events
per
interaction 500 to 1,000 oligos and thus interaction fragments are optimal.
The
genomic region then covered depends only on the resolution, i.e. average
spacing of
the 1st restriction enzyme within the genome.
In the case of a 1st and 2nd restriction enzyme we chose the oligos and
capture
arrays as follows: In the human case this was done for the H19/IGF2 region on
chromosome 11 from basepair position -1,110,650 to -3,216,350, i.e. a
2,105,700 bp
sized region and 525 oligos. In the mouse case this was done for the p-G lo b
in region
to on chromosome 7 from basepair position -109,876,350 to 111,966,600, i.e.
a
2,090,250 bp sized region and 800 oligos.
HRHTiC/C2 Regional DNA Sequencing Library Preparation - Microarray
Capturing
To produce a subselected regional T2C DNA fragment sequencing library from the
T2C whole genome DNA fragment sequencing library, the pooled DNA library after
ligation of the sequencing adapters was subject to subselection with the above
described newly and specifically developed capturing microarrays using the
NimbleGen Array capture protocol and hybridization system with enhancing
modifications from, NimbleGen Arrays User's Guide, Sequence Capture Array
Delivery version 3.2): The entire procedure consisted of i) microarray
hybridization, ii)
washing before iii) elution of the captured regional DNA library from the
microarray.
i) Therefore, 3 h before the capturing, the hybridization system was set to 42
C, a first
heat block was set to 95 C, and another one to 70 C, to equilibrate. Then the
hybridization mixture was prepaired by adding 300 pl of 1 mg/ml Cot-1 DNA to
the
pooled DNA library after ligation of the sequencing adapters. In the case of
using
multiplexed samples not only the 4 sets of material were pooled but also the
multiplexed samples were pooled. This saves microarray capacity and since the
amount of DNA to be captures is war of the saturation of the microarray this
leaves
room for multiplexing up to 10 to 100 samples depending on the DNA amount,
concentrations, and method to be used. Here multiplexing was only down by
pooling
2 different materials. Then the sample was dried in a SpeedVacTm at 60 C for
around
30 to 45 min, 11.2 pl of VVVR water was added for rehydration, vortexed and
centrifuged at maximum speed for 30 sec, before placement on the 70 C heat
block
for 10 min to fully solubilize the DNA. After a second vortexing and again
centrifugation at
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53
maximum speed for 30 sec, 18.5 pl of 2X SC hybridization buffer and SC
hybridization component A are added, followed again by vortexing and again
centrifugation at maximum speed for 30 sec. Then to denature the DNA the
sample
was placed on the 95 C heat block for 10 min before another centrifugation at
maximum speed for 30 sec. Thereafter, the sample was placed at 42 C and from
there immediately loaded on the microarray hybridization chamber (the complete
microarray system was prepared in parallel) and hybridized at 42 C for 64 h.
ii) To wash the captured regional T2C DNA library on the microarray first the
elution
chamber was assembled according to the NimbleGen array user guide. Therefore,
the microarray slide was removed from the 42 C NimbleGene hybridization system
and placed directly into the disassembly basin containing 100m1 of SC wash
buffer II
heated to 47.5 C. After ¨10 sec used for equilibration the mixer was peeled of
and
the slide was transferred to a second wash tube containing SC wash buffer II
at
47.5 C, the closed wash tube was inverted 10 times at a rate of 1 inversion
per
second. Then the slide was transferred to a new wash tube containing 32m1 of
stringent wash buffer at 47.5 C, and the closed tube was inverted 10 times at
a rate
of 1 inversion per second; before resting at 47.5 C for 5 min, and again
inverted 10
times at a rate of 1 inversion per second. Then the slide was again
transferred to a
new tube containing 32m1 of stringent wash buffer at 47.5 C, and the closed
tube was
inverted 10 times at a rate of 1 inversion per second, before resting at 47.5
C for 5
min, and again inverted 10 times at a rate of 1 inversion per second. Then the
slide
was again transferred to a new tube containing 32m1 of SC wash buffer 1 at
room
temperature, and the closed tube was inverted at a rate of 1 inversion per
second for
2 min. Then the slide was again transferred to a new tube containing 32m1 of
SC
wash buffer II at room temperature, and the closed tube was inverted at a rate
of 1
inversion per second for 1 min. Then the slide was again transferred to a new
tube
containing 32m1 of SC wash buffer III at room temperature, and the closed tube
was
inverted 10 times at a rate of 1 inversion per second.
iii) To elute the captured regional T2C DNA fragment sequencing library from
the
microarray the slide was transferred to the NimbleGen EU elution system at
room
temperature. Then ¨900 pl of 125 mM NaOH were added to the elution chamber
until
it is full, and incubated for 10 min. The eluted regional DNA fragment
sequencing
library was pipetted to a 1.5 ml tube and filled up to 900 pl of 125 mM NaOH,
followed
by division equally in two new tubes containing 516 pl of a well mixed
solution of 16 pl
20% acetic acid solution and 500 pl Qiagen Buffer PBI prepared beforehand in a
1.5m1 tube. Then the mixture was transferred to a single MinElute column on a
54
centrifuge to draw the solution through the column in several steps of 700 pl
each.
Then 750 pl buffer PE was put the column and centrifuged through. Then the
MinElute column was put into a 2m1 collection tube and centrifuged at maximum
speed for 1 min. to remove any residual buffer PE. The flow-through was
discarded,
before placement of the MinElute column in a clean 1.5m1 tube, 25 pl of buffer
EB
was added to the column, incubated for 1 min, and centrifuge at maximum speed
for
1 min.
T2C Amplification, Cluster Generation, and Paired-end High-Throughput
Sequencing
First for paired-end sequencing the T2C regional DNA fragment sequencing
library
was enriched for sequencing first by PCR using Phusion polymerase using 30 sec
at
98 C, 12 cycles of (10 sec at 98 C, 30 sec at 60 C, 30 sec at 72 C), 5 min at
72C
final extension. For each 1 pg of T2C regional DNA fragment library 5 pl of
the PCR
primer cocktail and 25 pl PCR master mix was added to the PCR plate. For
purification AMPure XP beads (Beckman Coulter) were used by adding 1.8 pl
AMPure XP beads per 1.0 pl of DNA. This was incubated at room temperature for
5
minutes, placed on the magnetic stand and incubated at room temperature for 5
minutes, and the supernatant was discarded without disturbing the beads. The
beads
were washed 2 times with freshly prepared 70% ethanol, placed at 37 C for 5
minutes
to let the beads dry. Then the beads were resuspended in 30 pl resuspension
bufferand incubated at room temperature for 5 minutes, placed on the magnetic
stand
for 5 minutes, and finally 50 pl supernatant was transferred to a new tube.
One
microliter was finally loaded on an Agilent Technologies 2100 bioanalyzer
using a
DNA 1000 assay to determine the quality of the purified digested DNA.
Cluster generation was performed according to the Illumine cBot User Guide,
part#15006165 RevE). Briefly, 1 pl of a 10 nM TruSeq DNA library stock DNA was
denatured with NaOH, diluted to 10 pM and hybridized onto the flowcell. The
hybridized fragments are sequentially amplified, linearized and end-blocked
according
to the Illumine Paired-end Sequencing user guide protocol. After hybridization
of the
sequencing primer, sequencing-by-synthesis was performed using the HiSeq 2000
sequencer with a 101 cycle protocol according to the instructions of the
manufacturer.
The sequenced fragments were denaturated with NaOH using the HiSeq 2000 and
the index-primer was hybridized onto the fragments. The index was sequenced
with a
7-cycle protocol. The fragments are denaturated with NAOH, sequentially
amplified,
linearized and end-blocked. After hybridization of the sequencing primer,
sequencing-
by-synthesis of the third read was performed
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using the HiSeq 2000 sequencer with a 101-cycle protocol.
HRHTiCIC2 Sequence Mapping and Classification
The raw sequence reads were checked for the existence of the first restriction
5 enzyme recognition sequence in the sequencing direction. The sequence
after the
first enzyme recognition site was removed. If the bases of the recognition
site after
the overhang were not unambiguously, the read was further trimmed by removing
all
the bases after the end of the overhang. Then these trimmed sequences were
aligned
using the Burrows-Wheeler Alignment (BWA) tool to the whole human genome
10 NCB136/hg18 assembly and to the mouse NCBI37/mm9 assembly. Therefore the
following default parameter set was used (with the value of the parameter in
brackets):
bwa am n [options] <prefix> <in.fq>
-n NUM max #diff (int) or missing prob under 0.02 err rate (float) [0.04]
-o INT maximum number or fraction of gap opens [1]
-e INT maximum number of gap extensions, -1 for disabling long gaps [-1]
-i INT do not put an indel within INT bp towards the ends [5]
-d INT maximum occurrences for extending a long deletion [10]
-I INT seed length [32]
-k INT maximum differences in the seed [2]
-m INT maximum entries in the queue [2000000]
-t INT number of threads [1]
-M INT mismatch penalty [3]
-0 INT gap open penalty [11]
-E INT gap extension penalty [4]
-R INT stop searching when there are >INT equally best hits [30]
-q INT quality threshold for read trimming down to 35bp [0]
4 FILE file to write output to instead of stdout
-B INT length of barcode
-L log-scaled gap penalty for long deletions
-N non-iterative mode: search for all n-difference hits (sl000w)
-I the input is in the IIlumina 1.3+ FASTQ-like format
-b the input read file is in the BAM format
-0 use single-end reads only (effective with -b)
56
-1 use the 1st read in a pair (effective with -b)
-2 use the 2nd read in a pair (effective with -b)
-Y filter Casava-filtered sequences
In case of using a second restriction enzyme (and thus not in the case of
sonication)
the unique sequences were then aligned in a second step to a masked genome,
excluding the sequence parts between second restriction enzymes and that did
not
contain a first enzyme recognition site. Finally, only those sequences were
paired
using the SAMtools to generate paired-end Binary Alignment/Map (BAM) files,
which
showed in both the whole and masked genome reference sequences a unique
alignment. Note: the alignements are unique, but nevertheless contain
mismatches
etc., which are either do to sequencing errors or hint a difference of our
cells/mice to
the reference genome. Unfortunately, there is also no way of distinguishing
false
positive or false negative alignments. Consequently, the resulting paired-end
sequences then contain the interaction information with an error rate
determined by
the error rate of sequencing, the quality of the reference sequence, and the
difference
the DNA sequence of our cells/mice to this reference genome. A rough estimate
of
the false positive and false negative results for unique sequences without
mismatches
at the end of this process using known error rates indicate the error to be
smaller than
1% after accumulation of errors and the reduction of errors due to our
procedure. This
can be also deducted from the reduction of sequence pairs from the initial raw
sequence throughout the entire process to the final result.
Various modifications and variations of the described methods and system of
the
invention will be apparent to those skilled in the art without departing from
the scope
and spirit of the invention. Although the invention has been described in
connection
with specific preferred embodiments, it should be understood that the
invention as
claimed should not be unduly limited to such specific embodiments. Indeed,
various
modifications of the described modes for carrying out the invention which are
obvious
to those skilled in molecular biology or related fields are intended to be
within the
scope of the following claims.
Date Recue/Date Received 2021-05-25
